TMPRSS6 iRNA COMPOSITIONS AND METHODS OF USE THEREOF

ABSTRACT

The invention relates to RNAi agents, e.g., double-stranded RNAi agents, targeting the TMPRSS6 gene, and methods of using such RNAi agents to inhibit expression of TMPRSS6 and methods of treating subjects having a TMPRSS6 associated disorder, e.g., an iron overload associated disorder, such as β-thalassemia or hemochromatosis.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/695,254, filed on Sep. 5, 2017, which is a continuation of U.S.patent application Ser. No. 14/947,025, filed on Nov. 20, 2015, now U.S.Pat. No. 9,783,806, issued on Oct. 10, 2017, which is a 35 § U.S.C.111(a) continuation application which claims the benefit of priority toPCT/US2014/039149, filed on May 22, 2014, which, in turn, claimspriority to U.S. Provisional Patent Application No. 61/826,178, filed onMay 22, 2013, and U.S. Provisional Patent Application No. 61/912,988,filed on Dec. 6, 2013. The entire contents of each of the foregoingpatent applications are hereby incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 30, 2018, isnamed 121301_00705_Seq_Listing.txt and is 449,740 bytes in size.

BACKGROUND OF THE INVENTION

TMPRSS6 (Transmembrane Protease, Serine 6) gene encodes TMPRSS6, alsoknown as matriptase-2, a type II serine protease. It is primarilyexpressed in the liver, although high levels of TMPRSS6 mRNA are alsofound in the kidney, with lower levels in the uterus and much smalleramounts detected in many other tissues (Ramsay et al., Haematologica(2009), 94(6), 840-849). TMPRSS6 plays a role in iron homeostatis bybinding and proteolytically degrading the hepcidin activator and BMPco-receptor HJV (hemojuvelin), which causes down-regulation of hepcidinlevels.

TMPRSS6 consists of a short N-terminal intracytoplasmic tail, a type IItransmembrane domain, a stem region composed of two extracellular CUB(complement factor Cls/Clr, urchin embryonic growth factor and BMP (bonemorphogenetic protein)) domains, three LDLR (low-density-lipoproteinreceptor class A) domains, and a C-terminal trypsin-like serine proteasedomain. There are also consensus sites for N-glycosylation in theextracellular domain, and a potential phosphorylation site in theintracytoplasmic tail region.

Numerous disorders can be associated with iron overload, a conditioncharacterized by increased levels of iron. Iron overload can result inexcess iron deposition in various tissues and can lead to tissue andorgan damage. Accordingly, methods for effective treatment of disordersassociated with iron overload are currently needed.

SUMMARY OF THE INVENTION

The present invention provides compositions comprising RNAi agents,e.g., double-stranded iRNA agents, targeting TMPRSS6. The presentinvention also provides methods using the compositions of the inventionfor inhibiting TMPRSS6 expression and for treating TMPRSS6 associateddisorders, e.g., iron overload associated disorders, such asthalassemia, e.g., β-thalassemia, or hemochromatosis.

Accordingly, in one aspect, the present invention provides RNAi agents,e.g., double-stranded RNAi agents, capable of inhibiting the expressionof TMPRSS6 (matriptase-2) in a cell, wherein the double stranded RNAiagent comprises a sense strand and an antisense strand forming adouble-stranded region, wherein the sense strand comprises at least 15contiguous nucleotides differing by no more than 3 nucleotides from anyone of the nucleotide sequences of SEQ ID NO:1, SEQ ID NO:2, or SEQ IDNO:3, SEQ ID NO:4, or SEQ ID NO:5, and the antisense strand comprises atleast 15 contiguous nucleotides differing by no more than 3 nucleotidesfrom any one of the nucleotide sequences of SEQ ID NO:6, SEQ ID NO:7, orSEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10,

wherein substantially all of the nucleotides of the sense strand andsubstantially all of the nucleotides of the antisense strand aremodified nucleotides, and

wherein the sense strand is conjugated to a ligand attached at the3′-terminus.

In one embodiment, all of the nucleotides of said sense strand and allof the nucleotides of said antisense strand are modified nucleotides.

In one embodiment, the sense strand and the antisense strand comprise aregion of complementarity which comprises at least 15 contiguousnucleotides differing by no more than 3 nucleotides from any one of theantisense sequences listed in any one of Tables 1, 2, 4, 5, 8, 10, and12.

In one embodiment, at least one of the modified nucleotides is selectedfrom the group consisting of a 3′-terminal deoxy-thymine (dT)nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modifiednucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, anabasic nucleotide, a 2′-amino-modified nucleotide, a 2′-alkyl-modifiednucleotide, a morpholino nucleotide, a phosphoramidate, a non-naturalbase comprising nucleotide, a nucleotide comprising a5′-phosphorothioate group, a nucleotide comprising a 5′ phosphate or 5′phosphate mimic (see, e.g., PCT Publication No. WO 2011/005860), and aterminal nucleotide linked to a cholesteryl derivative or a dodecanoicacid bisdecylamide group.

In one embodiment, at least one strand comprises a 3′ overhang of atleast 1 nucleotide. In another embodiment, at least one strand comprisesa 3′ overhang of at least 2 nucleotides.

In another aspect, the present invention provides RNAi agents, e.g.,double-stranded RNAi agents, capable of inhibiting the expression ofTMPRSS6 (matriptase-2) in a cell, wherein the double stranded RNAi agentcomprises a sense strand complementary to an antisense strand, whereinthe antisense strand comprises a region complementary to part of an mRNAencoding TMPRSS6, wherein each strand is about 14 to about 30nucleotides in length, wherein the double stranded RNAi agent isrepresented by formula (III):

sense: 5′n _(p)-N_(a)-(XXX)_(i)-N_(b)-YYY-N_(b)-(ZZZ)_(j)-N_(a)-n _(q)3′

antisense: 3′n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′-n_(q)′5′  (III)

wherein:

i, j, k, and l are each independently 0 or 1;

p, p′, q, and q′ are each independently 0-6;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 nucleotides which are either modified orunmodified or combinations thereof, each sequence comprising at leasttwo differently modified nucleotides;

-   -   each N_(b) and N_(b)′ independently represents an        oligonucleotide sequence comprising 0-10 nucleotides which are        either modified or unmodified or combinations thereof;    -   each n_(p), n_(p)′, n_(q), and n_(q)′, each of which may or may        not be present, independently represents an overhang nucleotide;    -   XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently        represent one motif of three identical modifications on three        consecutive nucleotides;    -   modifications on N_(b) differ from the modification on Y and        modifications on N_(b)′ differ from the modification on Y′; and    -   wherein the sense strand is conjugated to at least one ligand.

In one embodiment, i is 0; j is 0; i is 1; j is 1; both i and j are 0;or both i and j are 1. In another embodiment, k is 0; 1 is 0; k is 1; 1is 1; both k and l are 0; or both k and l are 1.

In one embodiment, XXX is complementary to X′X′X′, YYY is complementaryto Y′Y′Y′, and ZZZ is complementary to Z′Z′Z′.

In one embodiment, YYY motif occurs at or near the cleavage site of thesense strand.

In one embodiment, Y′Y′Y′ motif occurs at the 11, 12 and 13 positions ofthe antisense strand from the 5′-end.

In one embodiment, Y′ is 2′-O-methyl.

In one embodiment, formula (III) is represented by formula (IIIa):

sense: 5′n _(p)-N_(a)-YYY-N_(a)-n _(q)3′

antisense: 3′n _(p′)-N_(a)′-Y′Y′Y′-N_(a′)-n _(q′)5′  (IIIa).

In another embodiment, formula (III) is represented by formula (IIIb):

sense: 5′n _(p)-N_(a)-YYY-N_(b)-ZZZ-N_(a)-n _(q)3′

antisense: 3′n _(p′)-N_(a′)-Y′Y′Y′-N_(b′)-Z′Z′Z′-N_(a′)-n_(q′)5′  (IIIb)

wherein each N_(b) and N_(b)′ independently represents anoligonucleotide sequence comprising 1-5 modified nucleotides.

In yet another embodiment, formula (III) is represented by formula(IIIc):

sense: 5′n _(p)-N_(a)-XXX-N_(b)-YYY-N_(a)-n _(q)3′

antisense: 3′n _(p′)-N_(a′)-X′X′X′-N_(b′)-Y′Y′Y′-N_(a′)-n_(q′)5′  (IIIc)

wherein each N_(b) and N_(b)′ independently represents anoligonucleotide sequence comprising 1-5 modified nucleotides.

In one embodiment, formula (III) is represented by formula (IIId):

sense: 5′n _(p)-N_(a)-XXX-N_(b)-YYY-N_(b)-ZZZ-N_(a)-n _(q)3′

antisense: 3′n _(p′)-N_(a′)-X′X′X′-N_(b′)-Y′Y′Y′-N_(b′)-Z′Z′Z′-N_(a′)-n_(q′)5′  (IIId)

wherein each N_(b) and N_(b)′ independently represents anoligonucleotide sequence comprising 1-5 modified nucleotides and eachN_(a) and N_(a)′ independently represents an oligonucleotide sequencecomprising 2-10 modified nucleotides.

In one embodiment, the double-stranded region is 15-30 nucleotide pairsin length. In another embodiment, the double-stranded region is 17-23nucleotide pairs in length. In yet another embodiment, thedouble-stranded region is 17-25 nucleotide pairs in length. In oneembodiment, the double-stranded region is 23-27 nucleotide pairs inlength. In another embodiment, the double-stranded region is 19-21nucleotide pairs in length. In another embodiment, the double-strandedregion is 21-23 nucleotide pairs in length. In one embodiment, eachstrand has 15-30 nucleotides. In another embodiment, each strand has19-30 nucleotides.

In one embodiment, the modifications on the nucleotides are selectedfrom the group consisting of LNA, HNA, CeNA, 2′-methoxyethyl,2′-O-alkyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro, 2′-deoxy, 2′-hydroxyl,and combinations thereof. In another embodiment, the modifications onthe nucleotides are 2′-O-methyl or 2′-fluoro modifications.

In one embodiment, the ligand is one or more GalNAc derivatives attachedthrough a bivalent or trivalent branched linker. In another embodiment,the ligand is

In one embodiment, the ligand is attached to the 3′ end of the sensestrand.

In one embodiment, the RNAi agent is conjugated to the ligand as shownin the following schematic

wherein X is O or S. In a specific embodiment, X is O.

In one embodiment, the agent further comprises at least onephosphorothioate or methylphosphonate internucleotide linkage.

In one embodiment, the phosphorothioate or methylphosphonateinternucleotide linkage is at the 3′-terminus of one strand. In oneembodiment, the strand is the antisense strand. In another embodiment,the strand is the sense strand.

In one embodiment, the phosphorothioate or methylphosphonateinternucleotide linkage is at the 5′-terminus of one strand. In oneembodiment, the strand is the antisense strand. In another embodiment,the strand is the sense strand.

In one embodiment, the phosphorothioate or methylphosphonateinternucleotide linkage is at the both the 5′- and 3′-terminus of onestrand. In one embodiment, the strand is the antisense strand.

In one embodiment, the RNAi agent comprises 6-8 phosphorothioateinternucleotide linkages.

In one embodiment, the antisense strand comprises two phosphorothioateinternucleotide linkages at the 5′-terminus and two phosphorothioateinternucleotide linkages at the 3′-terminus, and the sense strandcomprises at least two phosphorothioate internucleotide linkages ateither the 5′-terminus or the 3′-terminus.

In one embodiment, the base pair at the 1 position of the 5′-end of theantisense strand of the duplex is an AU base pair.

In one embodiment, the Y nucleotides contain a 2′-fluoro modification.

In one embodiment, the Y′ nucleotides contain a 2′-O-methylmodification.

In one embodiment, p′>0. In another embodiment, p′=2.

In one embodiment, q′=0, p=0, q=0, and p′ overhang nucleotides arecomplementary to the target mRNA. In another embodiment, q′=0, p=0, q=0,and p′ overhang nucleotides are non-complementary to the target mRNA.

In one embodiment, the sense strand has a total of 21 nucleotides andthe antisense strand has a total of 23 nucleotides.

In one embodiment, at least one n_(p)′ is linked to a neighboringnucleotide via a phosphorothioate linkage.

In one embodiment, all n_(p)′ are linked to neighboring nucleotides viaphosphorothioate linkages.

In one embodiment, the RNAi agent is selected from the group of RNAiagents listed in any one of Tables 1, 2, 4, 5, 8, 10, and 12.

In one embodiment, the RNAi agent is AD-59743. In another embodiment,the RNAi agent is AD-60940.

In one aspect, the present invention provides double stranded RNAiagents for inhibiting expression of TMPRSS6 in a cell,

wherein the double stranded RNAi agent comprises a sense strand and anantisense strand forming a double stranded region,

wherein the sense strand comprises at least 15 contiguous nucleotidesdiffering by no more than 3 nucleotides from any one of the nucleotidesequences of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, SEQ ID NO:4, orSEQ ID NO:5, and the antisense strand comprises at least 15 contiguousnucleotides differing by no more than 3 nucleotides from any one of thenucleotide sequences of SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8, SEQ IDNO:9, or SEQ ID NO:10,

wherein substantially all of the nucleotides of the sense strandcomprise a modification selected from the group consisting of a2′-O-methyl modification and a 2′-fluoro modification,

wherein the sense strand comprises two phosphorothioate internucleotidelinkages at the 5′-terminus,

wherein substantially all of the nucleotides of the antisense strandcomprise a modification selected from the group consisting of a2′-O-methyl modification and a 2′-fluoro modification,

wherein the antisense strand comprises two phosphorothioateinternucleotide linkages at the 5′-terminus and two phosphorothioateinternucleotide linkages at the 3′-terminus, and

wherein the sense strand is conjugated to one or more GalNAc derivativesattached through a branched bivalent or trivalent linker at the3′-terminus.

In one embodiment, all of the nucleotides of the sense strand and all ofthe nucleotides of the antisense strand comprise a modification.

In another aspect, the present invention provides RNAi agents, e.g.,double stranded RNAi agents, capable of inhibiting the expression ofTMPRSS6 (matriptase-2) in a cell, wherein the double stranded RNAi agentcomprises a sense strand complementary to an antisense strand,

wherein the antisense strand comprises a region complementary to part ofan mRNA encoding TMPRSS6, wherein each strand is about 14 to about 30nucleotides in length, wherein the double stranded RNAi agent isrepresented by formula (III):

sense: 5′n _(p)-N_(a)-(XXX)_(i)-N_(b)-YYY-N_(b)-(ZZZ)_(j)-N_(a)-n _(q)3′

antisense: 3′n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′-n_(q)′5′  (III)

wherein:

i, j, k, and l are each independently 0 or 1;

p, p′, q, and q′ are each independently 0-6;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 nucleotides which are either modified orunmodified or combinations thereof, each sequence comprising at leasttwo differently modified nucleotides;

-   -   each N_(b) and N_(b)′ independently represents an        oligonucleotide sequence comprising 0-10 nucleotides which are        either modified or unmodified or combinations thereof;    -   each n_(p), n_(p)′, n_(q), and n_(q)′, each of which may or may        not be present independently represents an overhang nucleotide;    -   XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently        represent one motif of three identical modifications on three        consecutive nucleotides, and wherein the modifications are        2′-O-methyl or 2′-fluoro modifications;    -   modifications on N_(b) differ from the modification on Y and        modifications on N_(b)′ differ from the modification on Y′; and    -   wherein the sense strand is conjugated to at least one ligand.

In yet another aspect, the present invention provides RNAi agents, e.g.,double stranded RNAi agents, capable of inhibiting the expression ofTMPRSS6 (matriptase-2) in a cell, wherein the double stranded RNAi agentcomprises a sense strand complementary to an antisense strand, whereinthe antisense strand comprises a region complementary to part of an mRNAencoding TMPRSS6, wherein each strand is about 14 to about 30nucleotides in length, wherein the double stranded RNAi agent isrepresented by formula (III):

sense: 5′n _(p)-N_(a)-(XXX)_(i)-N_(b)-YYY-N_(b)-(ZZZ)_(j)-N_(a)-n _(q)3′

antisense: 3′n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′-n_(q)′5′  (III)

wherein:

i, j, k, and l are each independently 0 or 1;

each n_(p), n_(q), and n_(q)′, each of which may or may not be present,independently represents an overhang nucleotide;

p, q, and q′ are each independently 0-6;

n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotidevia a phosphorothioate linkage;

-   -   each N_(a) and N_(a)′ independently represents an        oligonucleotide sequence comprising 0-25 nucleotides which are        either modified or unmodified or combinations thereof, each        sequence comprising at least two differently modified        nucleotides;    -   each N_(b) and N_(b)′ independently represents an        oligonucleotide sequence comprising 0-10 nucleotides which are        either modified or unmodified or combinations thereof;        -   XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently            represent one motif of three identical modifications on            three consecutive nucleotides, and wherein the modifications            are 2′-O-methyl or 2′-fluoro modifications;    -   modifications on N_(b) differ from the modification on Y and        modifications on N_(b)′ differ from the modification on Y′; and    -   wherein the sense strand is conjugated to at least one ligand.

In a further aspect, the present invention provides RNAi agents, e.g.,double stranded RNAi agents, capable of inhibiting the expression ofTMPRSS6 (matriptase-2) in a cell, wherein the double stranded RNAi agentcomprises a sense strand complementary to an antisense strand,

wherein the antisense strand comprises a region complementary to part ofan mRNA encoding TMPRSS6, wherein each strand is about 14 to about 30nucleotides in length, wherein the double stranded RNAi agent isrepresented by formula (III):

sense: 5′n _(p)-N_(a)-(XXX)_(i)-N_(b)-YYY-N_(b)-(ZZZ)_(j)-N_(a)-n _(q)3′

antisense: 3′n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′-n_(q)′5′  (III)

wherein:

i, j, k, and l are each independently 0 or 1;

each n_(p), n_(q), and n_(q)′, each of which may or may not be present,independently represents an overhang nucleotide;

p, q, and q′ are each independently 0-6;

n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotidevia a phosphorothioate linkage;

-   -   each N_(a) and N_(a)′ independently represents an        oligonucleotide sequence comprising 0-25 nucleotides which are        either modified or unmodified or combinations thereof, each        sequence comprising at least two differently modified        nucleotides;    -   each N_(b) and N_(b)′ independently represents an        oligonucleotide sequence comprising 0-10 nucleotides which are        either modified or unmodified or combinations thereof;    -   XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently        represent one motif of three identical modifications on three        consecutive nucleotides, and wherein the modifications are        2′-O-methyl or 2′-fluoro modifications;    -   modifications on N_(b) differ from the modification on Y and        modifications on N_(b)′ differ from the modification on Y′; and    -   wherein the sense strand is conjugated to at least one ligand,        wherein the ligand is one or more GalNAc derivatives attached        through a bivalent or trivalent branched linker.

In another aspect, the present invention provides RNAi agents, e.g.,double stranded RNAi agents capable of inhibiting the expression ofTMPRSS6 (matriptase-2) in a cell, wherein the double stranded RNAi agentcomprises a sense strand complementary to an antisense strand,

wherein the antisense strand comprises a region complementary to part ofan mRNA encoding TMPRSS6, wherein each strand is about 14 to about 30nucleotides in length, wherein the double stranded RNAi agent isrepresented by formula (III):

sense: 5′n _(p)-N_(a)-(XXX)_(i)-N_(b)-YYY-N_(b)-(ZZZ)_(j)-N_(a)-n _(q)3′

antisense: 3′n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′-n_(q)′5′  (III)

wherein:

i, j, k, and l are each independently 0 or 1;

-   -   each n_(p), n_(q), and n_(q)′, each of which may or may not be        present, independently represents an overhang nucleotide;

p, q, and q′ are each independently 0-6;

n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotidevia a phosphorothioate linkage;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 nucleotides which are either modified orunmodified or combinations thereof, each sequence comprising at leasttwo differently modified nucleotides;

-   -   each N_(b) and N_(b)′ independently represents an        oligonucleotide sequence comprising 0-10 nucleotides which are        either modified or unmodified or combinations thereof;    -   XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently        represent one motif of three identical modifications on three        consecutive nucleotides, and wherein the modifications are        2′-O-methyl or 2′-fluoro modifications;    -   modifications on N_(b) differ from the modification on Y and        modifications on N_(b)′ differ from the modification on Y′;    -   wherein the sense strand comprises at least one phosphorothioate        linkage; and

wherein the sense strand is conjugated to at least one ligand, whereinthe ligand is one or more GalNAc derivatives attached through a bivalentor trivalent branched linker.

In yet another aspect, the present invention provides RNAi agents, e.g.,double stranded RNAi agents, capable of inhibiting the expression ofTMPRSS6 (matriptase-2) in a cell, wherein the double stranded RNAi agentcomprises a sense strand complementary to an antisense strand, whereinthe antisense strand comprises a region complementary to part of an mRNAencoding TMPRSS6, wherein each strand is about 14 to about 30nucleotides in length, wherein the double stranded RNAi agent isrepresented by formula (III):

sense: 5′n _(p)-N_(a)-YYY-N_(a)-n _(q)3′

antisense: 3′n _(p)′-N_(a)′-Y′Y′Y′-N_(a)′-n _(q)′5′  (IIIa)

wherein:

-   -   each n_(p), n_(q), and n_(q)′, each of which may or may not be        present, independently represents an overhang nucleotide;

p, q, and q′ are each independently 0-6;

n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotidevia a phosphorothioate linkage;

-   -   each N_(a) and N_(a)′ independently represents an        oligonucleotide sequence comprising 0-25 nucleotides which are        either modified or unmodified or combinations thereof, each        sequence comprising at least two differently modified        nucleotides;        -   YYY and Y′Y′Y′ each independently represent one motif of            three identical modifications on three consecutive            nucleotides, and wherein the modifications are 2′-O-methyl            or 2′-fluoro modifications;        -   wherein the sense strand comprises at least one            phosphorothioate linkage; and

wherein the sense strand is conjugated to at least one ligand, whereinthe ligand is one or more GalNAc derivatives attached through a bivalentor trivalent branched linker.

In one embodiment, the present invention provides RNAi agent selectedfrom the group of RNAi agents listed in any one of Tables 1, 2, 4, 5, 8,19, and 12.

In one aspect, the present invention provides compositions comprising amodified antisense polynucleotide agent, wherein the agent is capable ofinhibiting the expression of TMPRSS6 in a cell, and comprises a sequencecomplementary to a sense sequence selected from the group of thesequences listed in any one of Tables 1, 2, 4, 5, 8, 10, and 12, whereinthe polynucleotide is about 14 to about 30 nucleotides in length.

The present invention also provides cells, vectors, host cells, andpharmaceutical compositions comprising, e.g., the double stranded RNAiagents of the invention.

In some embodiments, the RNAi agent is administered using apharmaceutical composition.

In preferred embodiments, the RNAi agent is administered in a solution.In some such embodiments, the siRNA is administered in an unbufferedsolution. In one embodiment, the siRNA is administered in water. Inother embodiments, the siRNA is administered with a buffer solution,such as an acetate buffer, a citrate buffer, a prolamine buffer, acarbonate buffer, or a phosphate buffer or any combination thereof. Insome embodiments, the buffer solution is phosphate buffered saline(PBS).

In one embodiment, the pharmaceutical compositions further comprise alipid formulation. In one embodiment, the lipid formulation comprises aLNP, or XTC. In another embodiment, the lipid formulation comprises aMC3.

In one aspect, the present invention provides methods of inhibitingTMPRSS6 expression in a cell. The methods include contacting the cellwith an RNAi agent, e.g., a double stranded RNAi agent, or a modifiedantisense polynucleotide agent of the invention, or vector of theinvention, or a pharmaceutical composition of the invention; andmaintaining the cell produced in step (a) for a time sufficient toobtain degradation of the mRNA transcript of a TMPRSS6 gene, therebyinhibiting expression of the TMPRSS6 gene in the cell.

In one embodiment, the cell is within a subject.

In one embodiment, the subject is a human.

In one embodiment, the TMPRSS6 expression is inhibited by at least about30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,98%, or 100%.

In another embodiment, hepcidin gene expression is increased by at leastabout 1.5-fold, about 2-fold, about 3-fold, about 4-fold, or about5-fold.

In yet another embodiment, serum hepcidin concentration is increased byat least about 10%, about 25%, about 50%, about 100%, about 150%, about200%, about 250%, or about 300%.

In one embodiment, serum iron concentration is decreased by at leastabout 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about80%, about 90%, about 95%, about 98% or about 100%.

In another embodiment, a percent transferrin saturation is decreased byat least about 20%, about 30%, about 40%, about 50%, about 60%, about70%, about 80%, about 90%, about 95%, about 98% or about 100%.

In another aspect, the present invention provides methods of treating asubject having a disorder mediated by, or associated with, TMPRSS6expression. The methods include administering to the subject atherapeutically effective amount of an RNAi agent, e.g., a doublestranded RNAi agent, of the invention, or a modified antisensepolynucleotide agent of the invention, or a vector of the invention, ora pharmaceutical composition of the invention, thereby treating thesubject.

In one aspect, the present invention provides methods of treating asubject having a TMPRSS6-associated disorder. The methods includesubcutaneously administering to the subject a therapeutically effectiveamount of a double stranded RNAi agent,

wherein the double stranded RNAi agent comprises a sense strand and anantisense strand forming a double stranded region,

wherein the sense strand comprises at least 15 contiguous nucleotidesdiffering by no more than 3 nucleotides from any one of the nucleotidesequences of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, SEQ ID NO:4, orSEQ ID NO:5, and the antisense strand comprises at least 15 contiguousnucleotides differing by no more than 3 nucleotides from any one of thenucleotide sequences of SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8, SEQ IDNO:9, or SEQ ID NO:10,

wherein substantially all of the nucleotides of the antisense strandcomprise a modification selected from the group consisting of a2′-O-methyl modification and a 2′-fluoro modification,

wherein the antisense strand comprises two phosphorothioateinternucleotide linkages at the 5′-terminus and two phosphorothioateinternucleotide linkages at the 3′-terminus,

wherein substantially all of the nucleotides of the sense strandcomprise a modification selected from the group consisting of a2′-O-methyl modification and a 2′-fluoro modification,

wherein the sense strand comprises two phosphorothioate internucleotidelinkages at the 5′-terminus and,

wherein the sense strand is conjugated to one or more GalNAc derivativesattached through a branched bivalent or trivalent linker at the3′-terminus, thereby treating the subject. In one embodiment, all of thenucleotides of the sense strand and all of the nucleotides of theantisense strand comprise a modification.

In one embodiment, the subject is a human.

In one embodiment, the subject has a disorder associated with ironoverload, e.g., hereditary hemochromatosis, β-thalassemia (e.g.,β-thalassemia major and β-thalassemia intermiedia) erythropoieticporphyria, Parkinson's Disease, Alzheimer's Disease or Friedreich'sAtaxia.

In one embodiment, the RNAi agent, e.g., double stranded RNAi agent, isadministered at a dose of about 0.01 mg/kg to about 10 mg/kg, about 1mg/kg to about 10 mg/kg, about 2 mg/kg to about 10 mg/kg, about 3 mg/kgto about 10 mg/kg, about 4 mg/kg to about 10 mg/kg, about 5 mg/kg toabout 15 mg/kg, about 6 mg/kg to about 15 mg/kg, about 7 mg/kg to about15 mg/kg, about 8 mg/kg to about 15 mg/kg, about 9 mg/kg to about 15mg/kg, about 10 mg/kg to about 20 mg/kg, about 12 mg/kg to about 20mg/kg, about 13 mg/kg to about 20 mg/kg, about 14 mg/kg to about 20mg/kg, about 15 mg/kg to about 20 mg/kg, about 16 mg/kg to about 20mg/kg or about 18 mg/kg to about 20 mg/kg. In particular embodiments,the double stranded RNAi agent is administered at a dose of about 0.1mg/kg, about 1.0 mg/kg, or about 3.0 mg/kg.

In one embodiment, the RNAi agent, e.g., double stranded RNAi agent, isadministered subcutaneously or intravenously.

In one embodiment, the RNAi agent is administered in two or more doses.In a specific embodiment, the RNAi agent is administered at intervalsselected from the group consisting of once every about 12 hours, onceevery about 24 hours, once every about 48 hours, once every about 72hours, once every about 96 hours, once about every 7 days, or once aboutevery 14 days. In particular embodiments, the RNAi agent is administeredonce a week for up to 2 weeks, up to 3 weeks, up to 4 weeks, up to 5weeks, or longer.

In yet another aspect, the present invention provides methods oftreating an iron overload associated disorder in a subject. The methodsinclude administering to the subject a therapeutically effective amountof an RNAi agent, e.g., a double stranded RNAi agent, or the vector ofthe invention, thereby treating the subject.

In one embodiment, the iron overload associated disorder ishemochromatosis. In another embodiment, the iron overload associateddisorder is a thalassemia, e.g., β-thalassemia (e.g., β-thalassemiamajor and β-thalassemia intermiedia), or erythropoietic porphyria. Inyet another embodiment, the iron overload associated disorder is aneurological disease, e.g., Parkinson's Disease, Alzheimer's Disease orFriedreich's Ataxia.

In one embodiment, the subject is a primate or rodent. In anotherembodiment, the subject is a human.

In one embodiment, the RNAi agent, e.g., double stranded RNAi agent, isadministered at a dose of about 0.01 mg/kg to about 10 mg/kg, about 0.5mg/kg to about 50 mg/kg, about 10 mg/kg to about 30 mg/kg, about 10mg/kg to about 20 mg/kg, about 15 mg/kg to about 20 mg/kg, about 15mg/kg to about 25 mg/kg, about 15 mg/kg to about 30 mg/kg, or about 20mg/kg to about 30 mg/kg.

In one embodiment, the RNAi agent, e.g., double stranded RNAi agent, isadministered subcutaneously or intravenously.

In one embodiment, the RNAi agent is administered in two or more doses.In a specific embodiment, the RNAi agent is administered at intervalsselected from the group consisting of once every about 12 hours, onceevery about 24 hours, once every about 48 hours, once every about 72hours, once every about 96 hours, once about every 7 days, or once aboutevery 14 days.

In one embodiment, administering results in a decrease in iron levels,ferritin level and/or transferrin saturation level in the subject.

In one embodiment, the methods further comprise determining the ironlevel in the subject.

In one embodiment, the methods of the invention which includeadministering an iRNA agent of the invention (or pharmaceuticalcomposition of the invention) to a subject are practiced in combinationwith administration of additional pharmaceuticals and/or othertherapeutic methods. In one embodiment, the methods of the inventionfurther comprise administering an iron chelator, e.g., deferiprone,deferoxamine, and deferasirox, to a subject.

The present invention is further illustrated by the following detaileddescription and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing relative levels of TMPRSS6 mRNA in the liverof wild-type mice following administration of a single dose of 1 mg/kg,3 mg/kg or 10 mg/kg of the iRNA agent AD-59743.

FIG. 2 is a graph showing relative levels of hepcidin mRNA in the liverof wild-type mice following administration of a single dose of 1 mg/kg,3 mg/kg or 10 mg/kg of the iRNA agent AD-59743.

FIG. 3A is a graph showing the levels of hepatic TMPRSS6 mRNA in C57BL/6mice at various time points following a single subcutaneous injection ofAD-60940 at a dose of 0.3 mg/kg, 1.0 mg/kg or 3.0 mg/kg, or PBS alone(control). Each data point represents the mean value from three mice.The standard deviation of the mean is represented by error bars.

FIG. 3B is a graph showing the levels of hepatic hepcidin mRNA inC57BL/6 mice at various time points following a single subcutaneousinjection of AD-60940 at a dose of 0.3 mg/kg, 1.0 mg/kg or 3.0 mg/kg, orPBS alone (control). Each data point represents the mean value fromthree mice. The standard deviation of the mean is represented by errorbars.

FIG. 3C is a graph showing the levels of serum hepcidin in C57BL/6 miceat various time points following a single subcutaneous injection ofAD-60940 at a dose of 0.3 mg/kg, 1.0 mg/kg or 3.0 mg/kg, or PBS alone(control). Each data point represents the mean value from three mice.The standard deviation of the mean is represented by error bars.

FIG. 3D is a graph showing the levels of total serum iron in C57BL/6mice at various time points following a single subcutaneous injection ofAD-60940 at a dose of 0.3 mg/kg, 1.0 mg/kg or 3.0 mg/kg, or PBS alone(control). Each data point represents the mean value from three mice.The standard deviation of the mean is represented by error bars.

FIG. 3E is a graph showing the percent transferrin saturation (FIG. 3E)in C57BL/6 mice at various time points following a single subcutaneousinjection of AD-60940 at a dose of 0.3 mg/kg, 1.0 mg/kg or 3.0 mg/kg, orPBS alone (control). Each data point represents the mean value fromthree mice. The standard deviation of the mean is represented by errorbars.

FIG. 3F is a graph demonstrating the relative hepatic TMPRSS6 mRNAconcentration as a function of AD-60940 dose at 11 days followingadministration. Each data point represents the maximum suppression ofTMPRSS6 mRNA concentration observed at each dose level. The data werefit to the Hill equation.

FIG. 4A is a schematic depicting the administration regimen of one doseper week for three weeks followed by sacrifice of the mice at day 21.FIG. 4B is a graph showing the levels of hepatic TMPRSS6 mRNA, hepatichepcidin mRNA, and percent transferrin saturation in C57BL/6 miceadministered a subcutaneous injection of AD-60940 at a dose of 0.3mg/kg, 1.0 mg/kg, or PBS (control) according to the regimen shown inFIG. 4A. Each bar represents the mean value from three mice. Thestandard deviation of the mean is represented by error bars.

FIG. 4C demonstrates the relative hepatic TMPRSS6 mRNA concentration asa function of AD-60940 dose. The data were fit to the Hill equation.

FIG. 5A is a graph showing the relationship between serum hepcidinconcentration and relative TMPRSS6 mRNA levels.

FIG. 5B is a graph showing the relationship between percent transferrinsaturation and relative TMPRSS6 mRNA levels.

FIG. 5C is a graph showing the relationship between serum hepcidinconcentration and relative hepcidin mRNA levels.

FIG. 5D is a graph showing the relationship between percent transferrinsaturation and serum hepcidin concentration.

FIG. 6 is a graph showing relative levels of TMPRSS6 mRNA in the liverof C57BL/6 mice following administration of a single subcutaneous doseof 3 mg/kg of the indicated iRNA agent or PBS (control). The barsrepresent the mean from three mice and the error bars represent thestandard deviation of the mean.

FIG. 7 is a graph showing relative levels of TMPRSS6 mRNA in the liverof C57BL/6 mice following a subcutaneous dose of 0.3 mg/kg or 1.0 mg/kgof the indicated iRNA agent, or PBS (control), once a week for threeweeks. The bars represent the mean from three mice and the error barsrepresent the standard deviation of the mean.

FIG. 8 shows the nucleotide sequence of Homo sapiens TMPRSS6 (SEQ IDNO:1).

FIG. 9 shows the nucleotide sequence of Mus musculus TMPRSS6 (SEQ IDNO:2).

FIG. 10 shows the nucleotide sequence of Rattus norvegicus TMPRSS6 (SEQID NO:3).

FIG. 11 shows the nucleotide sequence of Macaca mulatta TMPRSS6 (SEQ IDNO:4).

FIG. 12 shows the nucleotide sequence of Macaca mulatta TMPRSS6 (SEQ IDNO:5).

FIG. 13 shows the reverse complement of SEQ ID NO:1 (SEQ ID NO:6).

FIG. 14 shows the reverse complement of SEQ ID NO:2 (SEQ ID NO:7).

FIG. 15 shows the reverse complement of SEQ ID NO:3 (SEQ ID NO:8).

FIG. 16 shows the reverse complement of SEQ ID NO:4 (SEQ ID NO:9).

FIG. 17 shows the reverse complement of SEQ ID NO:5 (SEQ ID NO:10).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions comprising RNAi agents,e.g., double-stranded iRNA agents, targeting TMPRSS6. The presentinvention also provides methods using the compositions of the inventionfor inhibiting TMPRSS6 expression and for treating TMPRSS6 associateddisorders, e.g., β-thalassemia or hemochromatosis.

TMPRSS6 plays an important role in iron homeostasis as an inhibitor ofHAMP gene expression. The HAMP gene encodes the liver hormone hepcidin,which is a central regulator of iron homeostasis. Hepcidin binds to theiron exporter protein ferroportin (FPN1), which is localized mainly onabsorptive enterocytes, hepatocytes and macrophages. Hepcidin binding tothe extracellular domain of ferroportin leads to the internalization anddegradation of ferroportin, thus decreasing the absorption of dietaryiron from the intestine, and the release of iron from macrophages andhepatocytes. HAMP gene expression can be stimulated in response to ironthrough Bone Morphogenetic Protein (BMP)/Sons of Mothers AgainstDecapentaplegic (SMAD)-dependent signal transduction cascade mediated bythe BMP-co-receptor hemojuvelin (HJV). The key role of TMPRSS6 in HAMPregulation is in the inhibition of BMP-mediated HAMP upregulation.TMPRSS6 inhibits BMP-mediated HAMP upregulation by cleaving the BMPco-receptor HJV, which is essential for BMP-mediated HAMP upregulation;thus preventing BMP signaling, SMAD translocation to the nucleus, andHAMP transcriptional activation.

Several human and mouse studies have confirmed the role of TMPRSS6 inHAMP regulation and iron homeostasis (Du et al. Science 2008, Vol. 320,pp 1088-1092; Folgueras et al. Blood 2008, Vol. 112, pp 2539-45).Studies have shown that loss of function mutations in TMPRSS6 can leadto the upregulation of hepcidin expression, causing an inherited irondeficiency anemia called iron refractory iron deficiency anemia (IRIDA)(Finberg. Seminars in Hematology 2009, Vol. 46, pp 378-86), which ischaracterized by elevated hepcidin levels, hypochromic microcyticanemia, low mean corpuscular volume (MCV), low transferrin saturation,poor absorption of oral iron, and incomplete response to parenteraliron. However, loss of function mutations in positive regulators of HAMP(e.g., BMP1, BMP4, and HFE) have been shown to downregulate hepcidinexpression and cause iron overload disorders (Milet et al. Am J Hum Gen2007, Vol. 81, pp 799-807; Finberg et al. Blood 2011, Vol. 117, pp4590-9). In the primary iron overload disorders, collectively calledhereditary hemochromatosis (HH), in anemias characterized by massiveineffective hematopoiesis, and in iron overload (secondaryhemochromatosis), such as β-thalassemia intermedia (TI), hepcidin levelsare low despite elevated serum iron concentrations and iron stores. Amouse model of β-thalassemia intermedia has demonstrated that the lossof TMPRSS6 expression leads to elevated levels of hepcidin (Finberg 2010Oral Presentation: “TMPRSS6, an inhibitor of Hepatic BMP/Smad Signaling,is required for Hepcidin Suppression and Iron Loading in a Mouse Modelof β-Thalassemia.” American Society of Hematology Annual Meeting 2010,Abstract No.: 164).

The present invention describes iRNA agents, compositions and methodsfor modulating the expression of a TMPRSS6 gene. In certain embodiments,expression of TMPRSS6 is reduced or inhibited using a TMPRSS6-specificiRNA agent, thereby leading to increase HAMP expression, and decreasedserum iron levels. Thus, inhibition of TMPRSS6 gene expression oractivity using the iRNA compositions featured in the invention can be auseful approach to therapies aimed at reducing the iron levels in asubject. Such inhibition can be useful for treating iron overloadassociated disorders, such as hemochromatosis or thalassemia, e.g.,β-thalassemia (e.g., β-thalassemia major and β-thalassemia intermiedia).

I. Definitions

In order that the present invention may be more readily understood,certain terms are first defined. In addition, it should be noted thatwhenever a value or range of values of a parameter are recited, it isintended that values and ranges intermediate to the recited values arealso intended to be part of this invention.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element, e.g., a plurality of elements.

The term “including” is used herein to mean, and is used interchangeablywith, the phrase “including but not limited to”.

The term “or” is used herein to mean, and is used interchangeably with,the term “and/or,” unless context clearly indicates otherwise.

As used herein, “TMPRSS6” refers to the type II plasma membrane serineprotease (TTSP) gene or protein. TMPRSS6 is also known as matriptase-2,IRIDA (iron refractory iron-deficiency anemia), transmembrane proteaseserine 6, type II transmembrane serine protease 6, and membrane-boundmosaic serine proteinase matriptase-2. TMPRSS6 is a serine protease TypeII transmembrane protein of approximately 899 amino acids in length.TMPRSS6 contains multiple domains, e.g., a short endo domain, atransmembrane domain, a sea urchin sperm protein/enteropeptidasedomain/agrin (SEA) domain, two complement factor/urchin embryonic growthfactor/BMP domains (CUB), three LDL-R class a domains (LDLa), and atrypsin-like serine protease domain with conserved His-Asp-Ser triad(HDS). The term “TMPRSS6” includes human TMPRSS6, the amino acid andnucleotide sequence of which may be found in, for example, GenBankAccession No. GI:56682967; mouse TMPRSS6, the amino acid and nucleotidesequence of which may be found in, for example, GenBank Accession No.GI:125656151; rat TMPRSS6, the amino acid and nucleotide sequence ofwhich may be found in, for example, GenBank Accession No. GI:194474097;rhesus TMPRSS6, the amino acid and nucleotide sequence of which may befound in, for example, GenBank Accession No. XM_001085203.2(GI:297260989) and XM_001085319.1 (GI:109094061). Additional examples ofAGT mRNA sequences are readily available using publicly availabledatabases, e.g., GenBank, UniProt, OMIM, and the Macaca genome projectweb site.

The term “TMPRSS6,” as used herein, also refers to naturally occurringDNA sequence variations of the TMPRSS6 gene, such as a single nucleotidepolymorphism (SNP) in the TMPRSS6 gene. Exemplary SNPs may be found inthe dbSNP database available at www.ncbi.nlmn.nih.gov/projects/SNP.

As used herein, “target sequence” refers to a contiguous portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof a TMPRSS6 gene, including mRNA that is a product of RNA processing ofa primary transcription product.

As used herein, the term “strand comprising a sequence” refers to anoligonucleotide comprising a chain of nucleotides that is described bythe sequence referred to using the standard nucleotide nomenclature.

“G,” “C,” “A” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, and uracil as a base, respectively.“T” and “dT” are used interchangeably herein and refer to adeoxyribonucleotide wherein the nucleobase is thymine, e.g.,deoxyribothymine, 2′-deoxythymidine or thymidine. However, it will beunderstood that the term “ribonucleotide” or “nucleotide” or“deoxyribonucleotide” can also refer to a modified nucleotide, asfurther detailed below, or a surrogate replacement moiety. The skilledperson is well aware that guanine, cytosine, adenine, and uracil may bereplaced by other moieties without substantially altering the basepairing properties of an oligonucleotide comprising a nucleotide bearingsuch replacement moiety. For example, without limitation, a nucleotidecomprising inosine as its base may base pair with nucleotides containingadenine, cytosine, or uracil. Hence, nucleotides containing uracil,guanine, or adenine may be replaced in the nucleotide sequences of theinvention by a nucleotide containing, for example, inosine. Sequencescomprising such replacement moieties are embodiments of the invention.

The terms “iRNA”, “RNAi agent,” “iRNA agent,”, “RNA interference agent”as used interchangeably herein, refer to an agent that contains RNA asthat term is defined herein, and which mediates the targeted cleavage ofan RNA transcript via an RNA-induced silencing complex (RISC) pathway.iRNA directs the sequence-specific degradation of mRNA through a processknown as RNA interference (RNAi). The iRNA modulates, e.g., inhibits,the expression of TMPRSS6 in a cell, e.g., a cell within a subject, suchas a mammalian subject.

In one embodiment, an RNAi agent of the invention includes a singlestranded RNA that interacts with a target RNA sequence, e.g., a TMPRSS6target mRNA sequence, to direct the cleavage of the target RNA. Withoutwishing to be bound by theory, it is believed that long double strandedRNA introduced into cells is broken down into siRNA by a Type IIIendonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485).Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23base pair short interfering RNAs with characteristic two base 3′overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs arethen incorporated into an RNA-induced silencing complex (RISC) where oneor more helicases unwind the siRNA duplex, enabling the complementaryantisense strand to guide target recognition (Nykanen, et al., (2001)Cell 107:309). Upon binding to the appropriate target mRNA, one or moreendonucleases within the RISC cleave the target to induce silencing(Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in one aspect theinvention relates to a single stranded RNA (siRNA) generated within acell and which promotes the formation of a RISC complex to effectsilencing of the target gene, i.e., a TMPRSS6 gene. Accordingly, theterm “siRNA” is also used herein to refer to an RNAi as described above.

In another embodiment, the RNAi agent may be a single-stranded siRNAthat is introduced into a cell or organism to inhibit a target mRNA.Single-stranded RNAi agents bind to the RISC endonuclease Argonaute 2,which then cleaves the target mRNA. The single-stranded siRNAs aregenerally 15-30 nucleotides and are chemically modified. The design andtesting of single-stranded siRNAs are described in U.S. Pat. No.8,101,348 and in Lima et al., (2012) Cell 150: 883-894, the entirecontents of each of which are hereby incorporated herein by reference.Any of the antisense nucleotide sequences described herein may be usedas a single-stranded siRNA as described herein or as chemically modifiedby the methods described in Lima et al., (2012) Cell 150:883-894.

In yet another embodiment, the present invention providessingle-stranded antisense oligonucleotide molecules targeting TMPRSS6. A“single-stranded antisense oligonucleotide molecule” is complementary toa sequence within the target mRNA (i.e., TMPRSS6). Single-strandedantisense oligonucleotide molecules can inhibit translation in astoichiometric manner by base pairing to the mRNA and physicallyobstructing the translation machinery, see Dias, N. et al., (2002) MolCancer Ther 1:347-355. Alternatively, the single-stranded antisenseoligonucleotide molecules inhibit a target mRNA by hydridizing to thetarget and cleaving the target through an RNaseH cleavage event. Thesingle-stranded antisense oligonucleotide molecule may be about 10 toabout 30 nucleotides in length and have a sequence that is complementaryto a target sequence. For example, the single-stranded antisenseoligonucleotide molecule may comprise a sequence that is at least about10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more contiguousnucleotides from any one of the antisense nucleotide sequences describedherein, e.g., the sequences provided in any one of Tables, 1, 2, 4, 5,8, 10, and 12, or bind any of the target sites described herein. Thesingle-stranded antisense oligonucleotide molecules may comprisemodified RNA, DNA, or a combination thereof.

In another embodiment, an “iRNA” for use in the compositions, uses, andmethods of the invention is a double-stranded RNA and is referred toherein as a “double stranded RNAi agent,” “double-stranded RNA (dsRNA)molecule,” “dsRNA agent,” or “dsRNA”. The term “dsRNA”, refers to acomplex of ribonucleic acid molecules, having a duplex structurecomprising two anti-parallel and substantially complementary nucleicacid strands, referred to as having “sense” and “antisense” orientationswith respect to a target RNA, i.e., a TMPRSS6 gene. In some embodimentsof the invention, a double-stranded RNA (dsRNA) triggers the degradationof a target RNA, e.g., an mRNA, through a post-transcriptionalgene-silencing mechanism referred to herein as RNA interference or RNAi.

In general, the majority of nucleotides of each strand of a dsRNAmolecule are ribonucleotides, but as described in detail herein, each orboth strands can also include one or more non-ribonucleotides, e.g., adeoxyribonucleotide and/or a modified nucleotide. In addition, as usedin this specification, an “RNAi agent” may include ribonucleotides withchemical modifications; an RNAi agent may include substantialmodifications at multiple nucleotides. Such modifications may includeall types of modifications disclosed herein or known in the art. Anysuch modifications, as used in a siRNA type molecule, are encompassed by“RNAi agent” for the purposes of this specification and claims.

The two strands forming the duplex structure may be different portionsof one larger RNA molecule, or they may be separate RNA molecules. Wherethe two strands are part of one larger molecule, and therefore areconnected by an uninterrupted chain of nucleotides between the 3′-end ofone strand and the 5′-end of the respective other strand forming theduplex structure, the connecting RNA chain is referred to as a “hairpinloop.” Where the two strands are connected covalently by means otherthan an uninterrupted chain of nucleotides between the 3′-end of onestrand and the 5′-end of the respective other strand forming the duplexstructure, the connecting structure is referred to as a “linker.” TheRNA strands may have the same or a different number of nucleotides. Themaximum number of base pairs is the number of nucleotides in theshortest strand of the dsRNA minus any overhangs that are present in theduplex. In addition to the duplex structure, an RNAi agent may compriseone or more nucleotide overhangs.

In one embodiment, an RNAi agent of the invention is a dsRNA of 24-30nucleotides that interacts with a target RNA sequence, e.g., a TMPRSS6target mRNA sequence, to direct the cleavage of the target RNA. Withoutwishing to be bound by theory, long double stranded RNA introduced intocells is broken down into siRNA by a Type III endonuclease known asDicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, aribonuclease-III-like enzyme, processes the dsRNA into 19-23 base pairshort interfering RNAs with characteristic two base 3′ overhangs(Bernstein, et al., (2001) Nature 409:363). The siRNAs are thenincorporated into an RNA-induced silencing complex (RISC) where one ormore helicases unwind the siRNA duplex, enabling the complementaryantisense strand to guide target recognition (Nykanen, et al., (2001)Cell 107:309). Upon binding to the appropriate target mRNA, one or moreendonucleases within the RISC cleave the target to induce silencing(Elbashir, et al., (2001) Genes Dev. 15:188).

As used herein, a “nucleotide overhang” refers to the unpairednucleotide or nucleotides that protrude from the duplex structure of anRNAi agent when a 3′-end of one strand of the RNAi agent extends beyondthe 5′-end of the other strand, or vice versa. “Blunt” or “blunt end”means that there are no unpaired nucleotides at that end of the doublestranded RNAi agent, i.e., no nucleotide overhang. A “blunt ended” RNAiagent is a dsRNA that is double-stranded over its entire length, i.e.,no nucleotide overhang at either end of the molecule. The RNAi agents ofthe invention include RNAi agents with nucleotide overhangs at one end(i.e., agents with one overhang and one blunt end) or with nucleotideoverhangs at both ends.

The term “antisense strand” refers to the strand of a double strandedRNAi agent which includes a region that is substantially complementaryto a target sequence (e.g., a human TMPRSS6 mRNA). As used herein, theterm “region complementary to part of an mRNA encoding transthyretin”refers to a region on the antisense strand that is substantiallycomplementary to part of a TMPRSS6 mRNA sequence. Where the region ofcomplementarity is not fully complementary to the target sequence, themismatches are most tolerated in the terminal regions and, if present,are generally in a terminal region or regions, e.g., within 6, 5, 4, 3,or 2 nucleotides of the 5′ and/or 3′ terminus.

The term “sense strand,” as used herein, refers to the strand of a dsRNAthat includes a region that is substantially complementary to a regionof the antisense strand.

As used herein, the term “cleavage region” refers to a region that islocated immediately adjacent to the cleavage site. The cleavage site isthe site on the target at which cleavage occurs. In some embodiments,the cleavage region comprises three bases on either end of, andimmediately adjacent to, the cleavage site. In some embodiments, thecleavage region comprises two bases on either end of, and immediatelyadjacent to, the cleavage site. In some embodiments, the cleavage sitespecifically occurs at the site bound by nucleotides 10 and 11 of theantisense strand, and the cleavage region comprises nucleotides 11, 12and 13.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of anoligonucleotide or polynucleotide comprising the first nucleotidesequence to hybridize and form a duplex structure under certainconditions with an oligonucleotide or polynucleotide comprising thesecond nucleotide sequence, as will be understood by the skilled person.Such conditions can, for example, be stringent conditions, wherestringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Otherconditions, such as physiologically relevant conditions as may beencountered inside an organism, can apply. For example, a complementarysequence is sufficient to allow the relevant function of the nucleicacid to proceed, e.g., RNAi. The skilled person will be able todetermine the set of conditions most appropriate for a test ofcomplementarity of two sequences in accordance with the ultimateapplication of the hybridized nucleotides.

Sequences can be “fully complementary” with respect to each when thereis base-pairing of the nucleotides of the first nucleotide sequence withthe nucleotides of the second nucleotide sequence over the entire lengthof the first and second nucleotide sequences. However, where a firstsequence is referred to as “substantially complementary” with respect toa second sequence herein, the two sequences can be fully complementary,or they may form one or more, but generally not more than 4, 3 or 2mismatched base pairs upon hybridization, while retaining the ability tohybridize under the conditions most relevant to their ultimateapplication. However, where two oligonucleotides are designed to form,upon hybridization, one or more single stranded overhangs, suchoverhangs shall not be regarded as mismatches with regard to thedetermination of complementarity. For example, a dsRNA comprising oneoligonucleotide 21 nucleotides in length and another oligonucleotide 23nucleotides in length, wherein the longer oligonucleotide comprises asequence of 21 nucleotides that is fully complementary to the shorteroligonucleotide, may yet be referred to as “fully complementary” for thepurposes described herein.

“Complementary” sequences, as used herein, may also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in as far as the aboverequirements with respect to their ability to hybridize are fulfilled.Such non-Watson-Crick base pairs includes, but not limited to, G:UWobble or Hoogstein base pairing.

The terms “complementary,” “fully complementary” and “substantiallycomplementary” herein may be used with respect to the base matchingbetween the sense strand and the antisense strand of a dsRNA, or betweenthe antisense strand of a dsRNA and a target sequence, as will beunderstood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary toat least part of” a messenger RNA (mRNA) refers to a polynucleotide thatis substantially complementary to a contiguous portion of the mRNA ofinterest (e.g., an mRNA encoding TMPRSS6) including a 5′ UTR, an openreading frame (ORF), or a 3′ UTR. For example, a polynucleotide iscomplementary to at least a part of a TMPRSS6 mRNA if the sequence issubstantially complementary to a non-interrupted portion of an mRNAencoding TMPRSS6.

The term “inhibiting,” as used herein, is used interchangeably with“reducing,” “silencing,” “downregulating,” “suppressing” and othersimilar terms, and includes any level of inhibition.

The phrase “inhibiting expression of a TMPRSS6,” as used herein,includes inhibition of expression of any TMPRSS6 gene (such as, e.g., amouse TMPRSS6 gene, a rat TMPRSS6 gene, a monkey TMPRSS6 gene, or ahuman TMPRSS6 gene) as well as variants, (e.g., naturally occurringvariants), or mutants of a TMPRSS6 gene. Thus, the TMPRSS6 gene may be awild-type TMPRSS6 gene, a mutant TMPRSS6 gene, or a transgenic TMPRSS6gene in the context of a genetically manipulated cell, group of cells,or organism.

“Inhibiting expression of a TMPRSS6 gene” includes any level ofinhibition of a TMPRSS6 gene, e.g., at least partial suppression of theexpression of a TMPRSS6 gene, such as an inhibition of at least about5%, at least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90%, at leastabout 91%, at least about 92%, at least about 93%, at least about 94%.at least about 95%, at least about 96%, at least about 97%, at leastabout 98%, or at least about 99%.

The expression of a TMPRSS6 gene may be assessed based on the level ofany variable associated with TMPRSS6 gene expression, e.g., TMPRSS6 mRNAlevel, TMPRSS6 protein level, hepcidin mRNA level, hepcidin proteinlevel, or iron levels in tissues or serum. Inhibition may be assessed bya decrease in an absolute or relative level of one or more of thesevariables compared with a control level. The control level may be anytype of control level that is utilized in the art, e.g., a pre-dosebaseline level, or a level determined from a similar subject, cell, orsample that is untreated or treated with a control (such as, e.g.,buffer only control or inactive agent control).

The phrase “contacting a cell with a double stranded RNAi agent,” asused herein, includes contacting a cell by any possible means.Contacting a cell with a double stranded RNAi agent includes contactinga cell in vitro with the RNAi agent or contacting a cell in vivo withthe RNAi agent. The contacting may be done directly or indirectly. Thus,for example, the RNAi agent may be put into physical contact with thecell by the individual performing the method, or alternatively, the RNAiagent may be put into a situation that will permit or cause it tosubsequently come into contact with the cell.

Contacting a cell in vitro may be done, for example, by incubating thecell with the RNAi agent. Contacting a cell in vivo may be done, forexample, by injecting the RNAi agent into or near the tissue where thecell is located, or by injecting the RNAi agent into another area, thebloodstream or the subcutaneous space, such that the agent willsubsequently reach the tissue where the cell to be contacted is located.For example, the RNAi agent may contain and/or be coupled to a ligand,e.g., a GalNAc3 ligand, that directs the RNAi agent to a site ofinterest, e.g., the liver. Combinations of in vitro and in vivo methodsof contacting are also possible. In connection with the methods of theinvention, a cell might also be contacted in vitro with an RNAi agentand subsequently transplanted into a subject.

A “patient” or “subject,” as used herein, is intended to include eithera human or non-human animal, preferably a mammal, e.g., human or amonkey. Most preferably, the subject or patient is a human.

A “TMPRSS6 associated disorder”, as used herein, is intended to includeany disorder that can be treated or prevented, or the symptoms of whichcan be alleviated, by inhibiting the expression of TMPRSS6. In someembodiments, the TMPRSS6 associated disorder is also associated withiron overload, a condition characterized by elevated iron levels, oriron dysregulation. Iron overload may be caused, for example, byhereditary conditions, by elevated iron uptake from diet, or by excessiron administered parenterally that includes intravenous injection ofexcess iron, and transfusional iron overload.

TMPRSS6 associated disorders include, but are not limited to, hereditaryhemochromatosis, idiopathic hemochromatosis, primary hemochromatosis,secondary hemochromatosis, severe juvenile hemochromatosis, neonatalhemochromatosis, sideroblastic anemia, hemolytic anemia,dyserythropoietic anemia, sickle-cell anemia, hemoglobinopathy,thalassemia (e.g., β-thalassemia and α-thalassemia), chronic liverdiseases, porphyria cutanea tarda, erythropoietic porphyria,atransferrinemia, hereditary tyrosinemia, cerebrohepatorenal syndrome,idiopathic pulmonary hemosiderosis, renal hemosiderosis.

TMPRSS6 associated disorders include disorders associated with oraladministration of excess iron, transfusional iron overload andintravenous injection of excess iron.

TMPRSS6 associated disorders also include disorders with symptoms thatare associated with or may be caused by iron overload. Such symptomsinclude increased risk for liver disease (cirrhosis, cancer), heartattack or heart failure, diabetes mellitus, osteoarthritis,osteoporosis, metabolic syndrome, hypothyroidism, hypogonadism, and insome cases premature death. In one embodiment, TMPRSS6 associateddisorders include neurodegenerative disorders associated with ironoverload and/or iron dysregulation, such as Alzheimer's Disease,Parkinson's Disease, Huntington's Disease, Friedreich's Ataxia, epilepsyand multiple sclerosis. Administration of an iRNA that targets TMPRSS6,e.g., an iRNA described in any one of Tables 1, 2, 4, 5, 8, 10, and 12can treat one or more of these symptoms, or prevent the development orprogression of a disease or disorder that is aggravated by increasediron levels.

In one embodiment, a TMPRSS6 associated disorder is a β-thalassemia. Aβ-thalassemia is any one of a group of hereditary disorderscharacterized by a genetic deficiency in the synthesis of beta-globinchains. In the homozygous state, beta thalassemia (“thalassemia major”)causes severe, transfusion-dependent anemia. In the heterozygous state,the beta thalassemia trait (“thalassemia minor”) causes mild to moderatemicrocytic anemia.

“Thalassemia intermedia” is a β-thalassemia that results in subjects inwhom the clinical severity of the disease is somewhere between the mildsymptoms of β-thalassemia minor and the β-thalassemia major. Thediagnosis is a clinical one that is based on the patient maintaining asatisfactory hemoglobin (Hb) level of at least 6-7 g/dL at the time ofdiagnosis without the need for regular blood transfusions.

In one embodiment, a β-thalassemia is thalassemia major. In anotherembodiment, a β-thalassemia is thalassemia intermedia.

“Therapeutically effective amount,” as used herein, is intended toinclude the amount of an RNAi agent that, when administered to a patientfor treating a TMPRSS6 associated disease, is sufficient to effecttreatment of the disease (e.g., by diminishing, ameliorating ormaintaining the existing disease or one or more symptoms of disease).The “therapeutically effective amount” may vary depending on the RNAiagent, how the agent is administered, the disease and its severity andthe history, age, weight, family history, genetic makeup, stage ofpathological processes mediated by TMPRSS6 expression, the types ofpreceding or concomitant treatments, if any, and other individualcharacteristics of the patient to be treated.

“Prophylactically effective amount,” as used herein, is intended toinclude the amount of an RNAi agent that, when administered to a subjectwho does not yet experience or display symptoms of a TMPRSS6-associateddisease, but who may be predisposed to the disease, is sufficient toprevent or ameliorate the disease or one or more symptoms of thedisease. Ameliorating the disease includes slowing the course of thedisease or reducing the severity of later-developing disease. The“prophylactically effective amount” may vary depending on the RNAiagent, how the agent is administered, the degree of risk of disease, andthe history, age, weight, family history, genetic makeup, the types ofpreceding or concomitant treatments, if any, and other individualcharacteristics of the patient to be treated.

A “therapeutically-effective amount” or “prophylacticaly effectiveamount” also includes an amount of an RNAi agent that produces somedesired local or systemic effect at a reasonable benefit/risk ratioapplicable to any treatment. RNAi gents employed in the methods of thepresent invention may be administered in a sufficient amount to producea reasonable benefit/risk ratio applicable to such treatment.

The term “sample,” as used herein, includes a collection of similarfluids, cells, or tissues isolated from a subject, as well as fluids,cells, or tissues present within a subject. Examples of biologicalfluids include blood, serum and serosal fluids, plasma, cerebrospinalfluid, ocular fluids, lymph, urine, saliva, and the like. Tissue samplesmay include samples from tissues, organs or localized regions. Forexample, samples may be derived from particular organs, parts of organs,or fluids or cells within those organs. In certain embodiments, samplesmay be derived from the liver (e.g., whole liver or certain segments ofliver or certain types of cells in the liver, such as, e.g.,hepatocytes). In preferred embodiments, a “sample derived from asubject” refers to blood or plasma drawn from the subject. In furtherembodiments, a “sample derived from a subject” refers to liver tissue(or subcomponents thereof) derived from the subject.

II. iRNAs of the Invention

Described herein are improved double-stranded RNAi agents which inhibitthe expression of a TMPRSS6 gene in a cell, such as a cell within asubject, e.g., a mammal, such as a human having a TMPRSS6 associateddisorder, e.g., β-thalassemia (e.g., β-thalassemia major andβ-thalassemia intermiedia) or hemochromatosis, and uses of suchdouble-stranded RNAi agents.

Accordingly, the invention provides double-stranded RNAi agents withchemical modifications capable of inhibiting the expression of a targetgene (i.e., a TMPRSS6 gene) in vivo. In certain aspects of theinvention, substantially all of the nucleotides of an iRNA of theinvention are modified. In other embodiments of the invention, all ofthe nucleotides of an iRNA of the invention are modified. iRNAs of theinvention in which “substantially all of the nucleotides are modified”are largely but not wholly modified and can include not more than 5, 4,3, 2, or 1 unmodified nucleotides.

The RNAi agent comprises a sense strand and an antisense strand. Eachstrand of the RNAi agent may range from 12-30 nucleotides in length. Forexample, each strand may be between 14-30 nucleotides in length, 17-30nucleotides in length, 19-30 nucleotides in length, 25-30 nucleotides inlength, 27-30 nucleotides in length, 17-23 nucleotides in length, 17-21nucleotides in length, 17-19 nucleotides in length, 19-25 nucleotides inlength, 19-23 nucleotides in length, 19-21 nucleotides in length, 21-25nucleotides in length, or 21-23 nucleotides in length.

The sense strand and antisense strand typically form a duplex doublestranded RNA (“dsRNA”), also referred to herein as an “RNAi agent.” Theduplex region of an RNAi agent may be 12-30 nucleotide pairs in length.For example, the duplex region can be between 14-30 nucleotide pairs inlength, 17-30 nucleotide pairs in length, 27-30 nucleotide pairs inlength, 17-23 nucleotide pairs in length, 17-21 nucleotide pairs inlength, 17-19 nucleotide pairs in length, 19-25 nucleotide pairs inlength, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs inlength, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs inlength. In another example, the duplex region is selected from 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides in length.

In one embodiment, the RNAi agent may contain one or more overhangregions and/or capping groups at the 3′-end, 5′-end, or both ends of oneor both strands. The overhang can be 1-6 nucleotides in length, forinstance 2-6 nucleotides in length, 1-5 nucleotides in length, 2-5nucleotides in length, 1-4 nucleotides in length, 2-4 nucleotides inlength, 1-3 nucleotides in length, 2-3 nucleotides in length, or 1-2nucleotides in length. The overhangs can be the result of one strandbeing longer than the other, or the result of two strands of the samelength being staggered. The overhang can form a mismatch with the targetmRNA or it can be complementary to the gene sequences being targeted orcan be another sequence. The first and second strands can also bejoined, e.g., by additional bases to form a hairpin, or by othernon-base linkers.

In one embodiment, the nucleotides in the overhang region of the RNAiagent can each independently be a modified or unmodified nucleotideincluding, but no limited to 2′-sugar modified, such as, 2-F,2′-O-methyl, thymidine (T), 2′-O-methoxyethyl-5-methyluridine (Teo),2′-O-methoxyethyladenosine (Aeo), 2′-O-methoxyethyl-5-methylcytidine(m5Ceo), and any combinations thereof. For example, TT can be anoverhang sequence for either end on either strand. The overhang can forma mismatch with the target mRNA or it can be complementary to the genesequences being targeted or can be another sequence.

The 5′- or 3′-overhangs at the sense strand, antisense strand or bothstrands of the RNAi agent may be phosphorylated. In some embodiments,the overhang region(s) contains two nucleotides having aphosphorothioate between the two nucleotides, where the two nucleotidescan be the same or different. In one embodiment, the overhang is presentat the 3′-end of the sense strand, antisense strand, or both strands. Inone embodiment, this 3′-overhang is present in the antisense strand. Inone embodiment, this 3′-overhang is present in the sense strand.

The RNAi agent may contain only a single overhang, which can strengthenthe interference activity of the RNAi, without affecting its overallstability. For example, the single-stranded overhang may be located atthe 3′-terminal end of the sense strand or, alternatively, at the3′-terminal end of the antisense strand. The RNAi may also have a bluntend, located at the 5′-end of the antisense strand (or the 3′-end of thesense strand) or vice versa. Generally, the antisense strand of the RNAihas a nucleotide overhang at the 3′-end, and the 5′-end is blunt. Whilenot wishing to be bound by theory, the asymmetric blunt end at the5′-end of the antisense strand and 3′-end overhang of the antisensestrand favor the guide strand loading into RISC process.

Any of the nucleic acids featured in the invention can be synthesizedand/or modified by methods well established in the art, such as thosedescribed in “Current protocols in nucleic acid chemistry,” Beaucage, S.L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, whichis hereby incorporated herein by reference. Modifications include, forexample, end modifications, e.g., 5′-end modifications (phosphorylation,conjugation, inverted linkages) or 3′-end modifications (conjugation,DNA nucleotides, inverted linkages, etc.); base modifications, e.g.,replacement with stabilizing bases, destabilizing bases, or bases thatbase pair with an expanded repertoire of partners, removal of bases(abasic nucleotides), or conjugated bases; sugar modifications (e.g., atthe 2′-position or 4′-position) or replacement of the sugar; and/orbackbone modifications, including modification or replacement of thephosphodiester linkages. Specific examples of iRNA compounds useful inthe embodiments described herein include, but are not limited to RNAscontaining modified backbones or no natural internucleoside linkages.RNAs having modified backbones include, among others, those that do nothave a phosphorus atom in the backbone. For the purposes of thisspecification, and as sometimes referenced in the art, modified RNAsthat do not have a phosphorus atom in their internucleoside backbone canalso be considered to be oligonucleosides. In some embodiments, amodified iRNA will have a phosphorus atom in its internucleosidebackbone.

Modified RNA backbones include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′-linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170;6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423;6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294;6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat.RE39464, the entire contents of each of which are hereby incorporatedherein by reference.

Modified RNA backbones that do not include a phosphorus atom thereinhave backbones that are formed by short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatoms and alkyl or cycloalkylinternucleoside linkages, or one or more short chain heteroatomic orheterocyclic internucleoside linkages. These include those havingmorpholino linkages (formed in part from the sugar portion of anucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

Representative U.S. patents that teach the preparation of the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and,5,677,439, the entire contents of each of which are hereby incorporatedherein by reference.

In other embodiments, suitable RNA mimetics are contemplated for use iniRNAs, in which both the sugar and the internucleoside linkage, i.e.,the backbone, of the nucleotide units are replaced with novel groups.The base units are maintained for hybridization with an appropriatenucleic acid target compound. One such oligomeric compound, an RNAmimetic that has been shown to have excellent hybridization properties,is referred to as a peptide nucleic acid (PNA). In PNA compounds, thesugar backbone of an RNA is replaced with an amide containing backbone,in particular an aminoethylglycine backbone. The nucleobases areretained and are bound directly or indirectly to aza nitrogen atoms ofthe amide portion of the backbone.

Representative U.S. patents that teach the preparation of PNA compoundsinclude, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331;and 5,719,262, the entire contents of each of which are herebyincorporated herein by reference. Additional PNA compounds suitable foruse in the iRNAs of the invention are described in, for example, inNielsen et al., Science, 1991, 254, 1497-1500.

Some embodiments featured in the invention include RNAs withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known asa methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of theabove-referenced U.S. Pat. No. 5,489,677, and the amide backbones of theabove-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAsfeatured herein have morpholino backbone structures of theabove-referenced U.S. Pat. No. 5,034,506.

Modified RNAs can also contain one or more substituted sugar moieties.The iRNAs, e.g., dsRNAs, featured herein can include one of thefollowing at the 2′-position: OH; F; O-, S-, or N-alkyl; O-, S-, orN-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl can be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Exemplary suitable modificationsinclude O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)._(n)OCH₃, O(CH₂)_(n)NH₂,O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where nand m are from 1 to about 10. In other embodiments, dsRNAs include oneof the following at the 2′ position: C₁ to C₁₀ lower alkyl, substitutedlower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN,Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of aniRNA, or a group for improving the pharmacodynamic properties of aniRNA, and other substituents having similar properties. In someembodiments, the modification includes a 2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martinet al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxygroup. Another exemplary modification is 2′-dimethylaminooxyethoxy,i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described inexamples herein below, and 2′-dimethylaminoethoxyethoxy (also known inthe art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂.

Other modifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications can alsobe made at other positions on the RNA of an iRNA, particularly the 3′position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linkeddsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs can alsohave sugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. Representative U.S. patents that teach thepreparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which arecommonly owned with the instant application. The entire contents of eachof the foregoing are hereby incorporated herein by reference.

An iRNA can also include nucleobase (often referred to in the art simplyas “base”) modifications or substitutions. As used herein, “unmodified”or “natural” nucleobases include the purine bases adenine (A) andguanine (G), and the pyrimidine bases thymine (T), cytosine (C) anduracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as deoxy-thymine (dT), 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo,particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat.No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry,Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; thosedisclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons,1990, these disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, YS., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke,S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobasesare particularly useful for increasing the binding affinity of theoligomeric compounds featured in the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications,CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of certain of theabove noted modified nucleobases as well as other modified nucleobasesinclude, but are not limited to, the above noted U.S. Pat. Nos.3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066;5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941;5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887;6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and7,495,088, the entire contents of each of which are hereby incorporatedherein by reference.

The RNA of an iRNA can also be modified to include one or more lockednucleic acids (LNA). A locked nucleic acid is a nucleotide having amodified ribose moiety in which the ribose moiety comprises an extrabridge connecting the 2′ and 4′ carbons. This structure effectively“locks” the ribose in the 3′-endo structural conformation. The additionof locked nucleic acids to siRNAs has been shown to increase siRNAstability in serum, and to reduce off-target effects (Elmen, J. et al.,(2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007)Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic AcidsResearch 31(12):3185-3193).

Representative U.S. patents that teach the preparation of locked nucleicacid nucleotides include, but are not limited to, the following: U.S.Pat. Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207;7,084,125; and 7,399,845, the entire contents of each of which arehereby incorporated herein by reference.

Potentially stabilizing modifications to the ends of RNA molecules caninclude N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc),N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol(Hyp-NHAc), thymidine-2′-0-deoxythymidine (ether),N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino),2-docosanoyl-uridine-3″-phosphate, inverted base dT(idT) and others.Disclosure of this modification can be found in PCT Publication No. WO2011/005861.

A. Modified iRNAs Comprising Motifs of the Invention

In certain aspects of the invention, the double-stranded RNAi agents ofthe invention include agents with chemical modifications as disclosed,for example, in U.S. Provisional Application No. 61/561,710, filed onNov. 18, 2011, or in PCT/US2012/065691, filed on Nov. 16, 2012, theentire contents of each of which are incorporated herein by reference.

As shown herein and in Provisional Application No. 61/561,710, asuperior result may be obtained by introducing one or more motifs ofthree identical modifications on three consecutive nucleotides into asense strand and/or antisense strand of a RNAi agent, particularly at ornear the cleavage site. In some embodiments, the sense strand andantisense strand of the RNAi agent may otherwise be completely modified.The introduction of these motifs interrupts the modification pattern, ifpresent, of the sense and/or antisense strand. The RNAi agent may beoptionally conjugated with a GalNAc derivative ligand, for instance onthe sense strand. The resulting RNAi agents present superior genesilencing activity.

More specifically, it has been surprisingly discovered that when thesense strand and antisense strand of the double-stranded RNAi agent aremodified to have one or more motifs of three identical modifications onthree consecutive nucleotides at or near the cleavage site of at leastone strand of an RNAi agent, the gene silencing activity of the RNAiagent was superiorly enhanced.

In one embodiment, the RNAi agent is a double ended bluntmer of 19nucleotides in length, wherein the sense strand contains at least onemotif of three 2′-F modifications on three consecutive nucleotides atpositions 7, 8, 9 from the 5′end. The antisense strand contains at leastone motif of three 2′-O-methyl modifications on three consecutivenucleotides at positions 11, 12, 13 from the 5′end.

In another embodiment, the RNAi agent is a double ended bluntmer of 20nucleotides in length, wherein the sense strand contains at least onemotif of three 2′-F modifications on three consecutive nucleotides atpositions 8, 9, 10 from the 5′end. The antisense strand contains atleast one motif of three 2′-O-methyl modifications on three consecutivenucleotides at positions 11, 12, 13 from the 5′end.

In yet another embodiment, the RNAi agent is a double ended bluntmer of21 nucleotides in length, wherein the sense strand contains at least onemotif of three 2′-F modifications on three consecutive nucleotides atpositions 9, 10, 11 from the 5′end. The antisense strand contains atleast one motif of three 2′-O-methyl modifications on three consecutivenucleotides at positions 11, 12, 13 from the 5′end.

In one embodiment, the RNAi agent comprises a 21 nucleotide sense strandand a 23 nucleotide antisense strand, wherein the sense strand containsat least one motif of three 2′-F modifications on three consecutivenucleotides at positions 9, 10, 11 from the 5′end; the antisense strandcontains at least one motif of three 2′-O-methyl modifications on threeconsecutive nucleotides at positions 11, 12, 13 from the 5′end, whereinone end of the RNAi agent is blunt, while the other end comprises a 2nucleotide overhang. Preferably, the 2 nucleotide overhang is at the3′-end of the antisense strand. When the 2 nucleotide overhang is at the3′-end of the antisense strand, there may be two phosphorothioateinternucleotide linkages between the terminal three nucleotides, whereintwo of the three nucleotides are the overhang nucleotides, and the thirdnucleotide is a paired nucleotide next to the overhang nucleotide. Inone embodiment, the RNAi agent additionally has two phosphorothioateinternucleotide linkages between the terminal three nucleotides at boththe 5′-end of the sense strand and at the 5′-end of the antisensestrand. In one embodiment, every nucleotide in the sense strand and theantisense strand of the RNAi agent, including the nucleotides that arepart of the motifs are modified nucleotides. In one embodiment eachresidue is independently modified with a 2′-O-methyl or 3′-fluoro, e.g.,in an alternating motif. Optionally, the RNAi agent further comprises aligand (preferably GalNAc₃).

In one embodiment, the RNAi agent comprises sense and antisense strands,wherein the RNAi agent comprises a first strand having a length which isat least 25 and at most 29 nucleotides and a second strand having alength which is at most 30 nucleotides with at least one motif of three2′-O-methyl modifications on three consecutive nucleotides at position11, 12, 13 from the 5′ end; wherein the 3′ end of the first strand andthe 5′ end of the second strand form a blunt end and the second strandis 1-4 nucleotides longer at its 3′ end than the first strand, whereinthe duplex region region which is at least 25 nucleotides in length, andthe second strand is sufficiently complementary to a target mRNA alongat least 19 nucleotide of the second strand length to reduce target geneexpression when the RNAi agent is introduced into a mammalian cell, andwherein dicer cleavage of the RNAi agent preferentially results in ansiRNA comprising the 3′ end of the second strand, thereby reducingexpression of the target gene in the mammal. Optionally, the RNAi agentfurther comprises a ligand.

In one embodiment, the sense strand of the RNAi agent contains at leastone motif of three identical modifications on three consecutivenucleotides, where one of the motifs occurs at the cleavage site in thesense strand.

In one embodiment, the antisense strand of the RNAi agent can alsocontain at least one motif of three identical modifications on threeconsecutive nucleotides, where one of the motifs occurs at or near thecleavage site in the antisense strand

For an RNAi agent having a duplex region of 17-23 nucleotide in length,the cleavage site of the antisense strand is typically around the 10, 11and 12 positions from the 5′-end. Thus the motifs of three identicalmodifications may occur at the 9, 10, 11 positions; 10, 11, 12positions; 11, 12, 13 positions; 12, 13, 14 positions; or 13, 14, 15positions of the antisense strand, the count starting from the 1^(st)nucleotide from the 5′-end of the antisense strand, or, the countstarting from the 1^(st) paired nucleotide within the duplex region fromthe 5′-end of the antisense strand. The cleavage site in the antisensestrand may also change according to the length of the duplex region ofthe RNAi from the 5′-end.

The sense strand of the RNAi agent may contain at least one motif ofthree identical modifications on three consecutive nucleotides at thecleavage site of the strand; and the antisense strand may have at leastone motif of three identical modifications on three consecutivenucleotides at or near the cleavage site of the strand. When the sensestrand and the antisense strand form a dsRNA duplex, the sense strandand the antisense strand can be so aligned that one motif of the threenucleotides on the sense strand and one motif of the three nucleotideson the antisense strand have at least one nucleotide overlap, i.e., atleast one of the three nucleotides of the motif in the sense strandforms a base pair with at least one of the three nucleotides of themotif in the antisense strand. Alternatively, at least two nucleotidesmay overlap, or all three nucleotides may overlap.

In one embodiment, the sense strand of the RNAi agent may contain morethan one motif of three identical modifications on three consecutivenucleotides. The first motif may occur at or near the cleavage site ofthe strand and the other motifs may be a wing modification. The term“wing modification” herein refers to a motif occurring at anotherportion of the strand that is separated from the motif at or near thecleavage site of the same strand. The wing modification is eitheradjacent to the first motif or is separated by at least one or morenucleotides. When the motifs are immediately adjacent to each other thenthe chemistry of the motifs are distinct from each other and when themotifs are separated by one or more nucleotide than the chemistries canbe the same or different. Two or more wing modifications may be present.For instance, when two wing modifications are present, each wingmodification may occur at one end relative to the first motif which isat or near cleavage site or on either side of the lead motif.

Like the sense strand, the antisense strand of the RNAi agent maycontain more than one motifs of three identical modifications on threeconsecutive nucleotides, with at least one of the motifs occurring at ornear the cleavage site of the strand. This antisense strand may alsocontain one or more wing modifications in an alignment similar to thewing modifications that may be present on the sense strand.

In one embodiment, the wing modification on the sense strand orantisense strand of the RNAi agent typically does not include the firstone or two terminal nucleotides at the 3′-end, 5′-end or both ends ofthe strand.

In another embodiment, the wing modification on the sense strand orantisense strand of the RNAi agent typically does not include the firstone or two paired nucleotides within the duplex region at the 3′-end,5′-end or both ends of the strand.

When the sense strand and the antisense strand of the RNAi agent eachcontain at least one wing modification, the wing modifications may fallon the same end of the duplex region, and have an overlap of one, two orthree nucleotides.

When the sense strand and the antisense strand of the RNAi agent eachcontain at least two wing modifications, the sense strand and theantisense strand can be so aligned that two modifications each from onestrand fall on one end of the duplex region, having an overlap of one,two or three nucleotides; two modifications each from one strand fall onthe other end of the duplex region, having an overlap of one, two orthree nucleotides; two modifications one strand fall on each side of thelead motif, having an overlap of one, two or three nucleotides in theduplex region.

In one embodiment, every nucleotide in the sense strand and antisensestrand of the RNAi agent, including the nucleotides that are part of themotifs, may be modified. Each nucleotide may be modified with the sameor different modification which can include one or more alteration ofone or both of the non-linking phosphate oxygens and/or of one or moreof the linking phosphate oxygens; alteration of a constituent of theribose sugar, e.g., of the 2′ hydroxyl on the ribose sugar; wholesalereplacement of the phosphate moiety with “dephospho” linkers;modification or replacement of a naturally occurring base; andreplacement or modification of the ribose-phosphate backbone.

As nucleic acids are polymers of subunits, many of the modificationsoccur at a position which is repeated within a nucleic acid, e.g., amodification of a base, or a phosphate moiety, or a non-linking O of aphosphate moiety. In some cases the modification will occur at all ofthe subject positions in the nucleic acid but in many cases it will not.By way of example, a modification may only occur at a 3′ or 5′ terminalposition, may only occur in a terminal region, e.g., at a position on aterminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of astrand. A modification may occur in a double strand region, a singlestrand region, or in both. A modification may occur only in the doublestrand region of a RNA or may only occur in a single strand region of aRNA. For example, a phosphorothioate modification at a non-linking Oposition may only occur at one or both termini, may only occur in aterminal region, e.g., at a position on a terminal nucleotide or in thelast 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in doublestrand and single strand regions, particularly at termini. The 5′ end orends can be phosphorylated.

It may be possible, e.g., to enhance stability, to include particularbases in overhangs, or to include modified nucleotides or nucleotidesurrogates, in single strand overhangs, e.g., in a 5′ or 3′ overhang, orin both. For example, it can be desirable to include purine nucleotidesin overhangs. In some embodiments all or some of the bases in a 3′ or 5′overhang may be modified, e.g., with a modification described herein.Modifications can include, e.g., the use of modifications at the 2′position of the ribose sugar with modifications that are known in theart, e.g., the use of deoxyribonucleotides, 2′-deoxy-2′-fluoro (2′-F) or2′-O-methyl modified instead of the ribosugar of the nucleobase, andmodifications in the phosphate group, e.g., phosphorothioatemodifications. Overhangs need not be homologous with the targetsequence.

In one embodiment, each residue of the sense strand and antisense strandis independently modified with LNA, HNA, CeNA, 2′-methoxyethyl,2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-deoxy, 2′-hydroxyl, or2′-fluoro. The strands can contain more than one modification. In oneembodiment, each residue of the sense strand and antisense strand isindependently modified with 2′-O-methyl or 2′-fluoro.

At least two different modifications are typically present on the sensestrand and antisense strand. Those two modifications may be the2′-O-methyl or 2′-fluoro modifications, or others.

In one embodiment, the N_(a) and/or N_(b) comprise modifications of analternating pattern. The term “alternating motif” as used herein refersto a motif having one or more modifications, each modification occurringon alternating nucleotides of one strand. The alternating nucleotide mayrefer to one per every other nucleotide or one per every threenucleotides, or a similar pattern. For example, if A, B and C eachrepresent one type of modification to the nucleotide, the alternatingmotif can be “ABABABABABAB . . . ,” “AABBAABBAABB . . . ,” “AABAABAABAAB. . . ,” “AAABAAABAAAB . . . ,” “AAABBBAAABBB . . . ,” or “ABCABCABCABC. . . ,” etc.

The type of modifications contained in the alternating motif may be thesame or different. For example, if A, B, C, D each represent one type ofmodification on the nucleotide, the alternating pattern, i.e.,modifications on every other nucleotide, may be the same, but each ofthe sense strand or antisense strand can be selected from severalpossibilities of modifications within the alternating motif such as“ABABAB . . . ”, “ACACAC . . . ” “BDBDBD . . . ” or “CDCDCD . . . ,”etc.

In one embodiment, the RNAi agent of the invention comprises themodification pattern for the alternating motif on the sense strandrelative to the modification pattern for the alternating motif on theantisense strand is shifted. The shift may be such that the modifiedgroup of nucleotides of the sense strand corresponds to a differentlymodified group of nucleotides of the antisense strand and vice versa.For example, the sense strand when paired with the antisense strand inthe dsRNA duplex, the alternating motif in the sense strand may startwith “ABABAB” from 5′-3′ of the strand and the alternating motif in theantisense strand may start with “BABABA” from 5′-3′ of the strand withinthe duplex region. As another example, the alternating motif in thesense strand may start with “AABBAABB” from 5′-3′ of the strand and thealternating motif in the antisenese strand may start with “BBAABBAA”from 5′-3′ of the strand within the duplex region, so that there is acomplete or partial shift of the modification patterns between the sensestrand and the antisense strand.

In one embodiment, the RNAi agent comprises the pattern of thealternating motif of 2′-O-methyl modification and 2′-F modification onthe sense strand initially has a shift relative to the pattern of thealternating motif of 2′-O-methyl modification and 2′-F modification onthe antisense strand initially, i.e., the 2′-O-methyl modifiednucleotide on the sense strand base pairs with a 2′-F modifiednucleotide on the antisense strand and vice versa. The 1 position of thesense strand may start with the 2′-F modification, and the 1 position ofthe antisense strand may start with the 2′-O-methyl modification.

The introduction of one or more motifs of three identical modificationson three consecutive nucleotides to the sense strand and/or antisensestrand interrupts the initial modification pattern present in the sensestrand and/or antisense strand. This interruption of the modificationpattern of the sense and/or antisense strand by introducing one or moremotifs of three identical modifications on three consecutive nucleotidesto the sense and/or antisense strand surprisingly enhances the genesilencing activity to the target gene.

In one embodiment, when the motif of three identical modifications onthree consecutive nucleotides is introduced to any of the strands, themodification of the nucleotide next to the motif is a differentmodification than the modification of the motif. For example, theportion of the sequence containing the motif is “ . . . N_(a)YYYN_(b) .. . ,” where “Y” represents the modification of the motif of threeidentical modifications on three consecutive nucleotide, and “N_(a)” and“N_(b)” represent a modification to the nucleotide next to the motif“YYY” that is different than the modification of Y, and where N_(a) andN_(b) can be the same or different modifications. Alternatively, N_(a)and/or N_(b) may be present or absent when there is a wing modificationpresent.

The RNAi agent may further comprise at least one phosphorothioate ormethylphosphonate internucleotide linkage. The phosphorothioate ormethylphosphonate internucleotide linkage modification may occur on anynucleotide of the sense strand or antisense strand or both strands inany position of the strand. For instance, the internucleotide linkagemodification may occur on every nucleotide on the sense strand and/orantisense strand; each internucleotide linkage modification may occur inan alternating pattern on the sense strand and/or antisense strand; orthe sense strand or antisense strand may contain both internucleotidelinkage modifications in an alternating pattern. The alternating patternof the internucleotide linkage modification on the sense strand may bethe same or different from the antisense strand, and the alternatingpattern of the internucleotide linkage modification on the sense strandmay have a shift relative to the alternating pattern of theinternucleotide linkage modification on the antisense strand.

In one embodiment, the RNAi comprises a phosphorothioate ormethylphosphonate internucleotide linkage modification in the overhangregion. For example, the overhang region may contain two nucleotideshaving a phosphorothioate or methylphosphonate internucleotide linkagebetween the two nucleotides. Internucleotide linkage modifications alsomay be made to link the overhang nucleotides with the terminal pairednucleotides within the duplex region. For example, at least 2, 3, 4, orall the overhang nucleotides may be linked through phosphorothioate ormethylphosphonate internucleotide linkage, and optionally, there may beadditional phosphorothioate or methylphosphonate internucleotidelinkages linking the overhang nucleotide with a paired nucleotide thatis next to the overhang nucleotide. For instance, there may be at leasttwo phosphorothioate internucleotide linkages between the terminal threenucleotides, in which two of the three nucleotides are overhangnucleotides, and the third is a paired nucleotide next to the overhangnucleotide. These terminal three nucleotides may be at the 3′-end of theantisense strand, the 3′-end of the sense strand, the 5′-end of theantisense strand, and/or the 5′end of the antisense strand.

In one embodiment, the 2 nucleotide overhang is at the 3′-end of theantisense strand, and there are two phosphorothioate internucleotidelinkages between the terminal three nucleotides, wherein two of thethree nucleotides are the overhang nucleotides, and the third nucleotideis a paired nucleotide next to the overhang nucleotide. Optionally, theRNAi agent may additionally have two phosphorothioate internucleotidelinkages between the terminal three nucleotides at both the 5′-end ofthe sense strand and at the 5′-end of the antisense strand.

In one embodiment, the RNAi agent comprises mismatch(es) with thetarget, within the duplex, or combinations thereof. The mismatch mayoccur in the overhang region or the duplex region. The base pair may beranked on the basis of their propensity to promote dissociation ormelting (e.g., on the free energy of association or dissociation of aparticular pairing, the simplest approach is to examine the pairs on anindividual pair basis, though next neighbor or similar analysis can alsobe used). In terms of promoting dissociation: A:U is preferred over G:C;G:U is preferred over G:C; and I:C is preferred over G:C (I=inosine).Mismatches, e.g., non-canonical or other than canonical pairings (asdescribed elsewhere herein) are preferred over canonical (A:T, A:U, G:C)pairings; and pairings which include a universal base are preferred overcanonical pairings.

In one embodiment, the RNAi agent comprises at least one of the first 1,2, 3, 4, or 5 base pairs within the duplex regions from the 5′-end ofthe antisense strand independently selected from the group of: A:U, G:U,I:C, and mismatched pairs, e.g., non-canonical or other than canonicalpairings or pairings which include a universal base, to promote thedissociation of the antisense strand at the 5′-end of the duplex.

In one embodiment, the nucleotide at the 1 position within the duplexregion from the 5′-end in the antisense strand is selected from thegroup consisting of A, dA, dU, U, and dT. Alternatively, at least one ofthe first 1, 2 or 3 base pair within the duplex region from the 5′-endof the antisense strand is an AU base pair. For example, the first basepair within the duplex region from the 5′-end of the antisense strand isan AU base pair.

In one embodiment, the sense strand sequence may be represented byformula (I):

5′n _(p)-N_(a)-(XXX)_(i)-N_(b)-YYY-N_(b)-(ZZZ)_(j)-N_(a)-n _(q)3′  (I)

wherein:

i and j are each independently 0 or 1;

p and q are each independently 0-6;

each N_(a) independently represents an oligonucleotide sequencecomprising 0-25 modified nucleotides, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b) independently represents an oligonucleotide sequencecomprising 0-10 modified nucleotides;

each n_(p) and n_(q) independently represent an overhang nucleotide;

wherein Nb and Y do not have the same modification; and

XXX, YYY and ZZZ each independently represent one motif of threeidentical modifications on three consecutive nucleotides. Preferably YYYis all 2′-F modified nucleotides.

In one embodiment, the N_(a) and/or N_(b) comprise modifications ofalternating pattern.

In one embodiment, the YYY motif occurs at or near the cleavage site ofthe sense strand. For example, when the RNAi agent has a duplex regionof 17-23 nucleotides in length, the YYY motif can occur at or thevicinity of the cleavage site (e.g.: can occur at positions 6, 7, 8, 7,8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12 or 11, 12, 13) of—the sensestrand, the count starting from the 1^(st) nucleotide, from the 5′-end;or optionally, the count starting at the 1^(st) paired nucleotide withinthe duplex region, from the 5′-end.

In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both iand j are 1. The sense strand can therefore be represented by thefollowing formulas:

5′n _(p)-N_(a)-YYY-N_(b)-ZZZ-N_(a)-n _(q)3′  (Ib);

5′n _(p)-N_(a)-XXX-N_(b)-YYY-N_(a)-n _(q)3′  (Ic); or

5′n _(p)-N_(a)-XXX-N_(b)-YYY-N_(b)-ZZZ-N_(a)-n _(q)3′  (Id).

When the sense strand is represented by formula (Ib), N_(b) representsan oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0modified nucleotides. Each N_(a) independently can represent anoligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

When the sense strand is represented as formula (Ic), N_(b) representsan oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4,0-2 or 0 modified nucleotides. Each N_(a) can independently represent anoligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

When the sense strand is represented as formula (Id), each N_(b)independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Preferably, N_(b) is 0, 1,2, 3, 4, 5 or 6 Each N_(a) can independently represent anoligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

Each of X, Y and Z may be the same or different from each other.

In other embodiments, i is 0 and j is 0, and the sense strand may berepresented by the formula:

5′n _(p)-N_(a)-YYY-N_(a)-n _(q)3′  (Ia).

When the sense strand is represented by formula (Ia), each N_(a)independently can represent an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

In one embodiment, the antisense strand sequence of the RNAi may berepresented by formula (II):

5′n _(q′)-N_(a)′-(Z′Z′Z′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(X′X′X′)_(l)-N′_(a)-n_(p)′3′  (II)

wherein:

k and l are each independently 0 or 1;

p′ and q′ are each independently 0-6;

each N_(a)′ independently represents an oligonucleotide sequencecomprising 0-25 modified nucleotides, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b)′ independently represents an oligonucleotide sequencecomprising 0-10 modified nucleotides;

each n_(p)′ and n_(q)′ independently represent an overhang nucleotide;

wherein N_(b)′ and Y′ do not have the same modification;

and

X′X′X′, Y′Y′Y′ and Z′Z′Z′ each independently represent one motif ofthree identical modifications on three consecutive nucleotides.

In one embodiment, the N_(a)′ and/or N_(b)′ comprise modifications ofalternating pattern.

The Y′Y′Y′ motif occurs at or near the cleavage site of the antisensestrand. For example, when the RNAi agent has a duplex region of 17-23nucleotide in length, the Y′Y′Y′ motif can occur at positions 9, 10, 11;10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisensestrand, with the count starting from the 1^(st) nucleotide, from the5′-end; or optionally, the count starting at the 1^(st) pairednucleotide within the duplex region, from the 5′-end. Preferably, theY′Y′Y′ motif occurs at positions 11, 12, 13.

In one embodiment, Y′Y′Y′ motif is all 2′-OMe modified nucleotides.

In one embodiment, k is 1 and 1 is 0, or k is 0 and 1 is 1, or both kand l are 1.

The antisense strand can therefore be represented by the followingformulas:

5′n _(q′)-N_(a)′-Z′Z′Z′-N_(b)′-Y′Y′Y′-N_(a)′-n _(p′)3′  (IIb);

5′n _(q′)-N_(a)′-Y′Y′Y′-N_(b)′-X′X′X′-n _(p′)3′  (IIc); or

5′n _(q′)-N_(a)′-Z′Z′Z′-N_(b)′-Y′Y′Y′-N_(b)′-X′X′X′-N_(a)′-n_(p′)3′  (IId).

When the antisense strand is represented by formula (IIb), N_(b)′represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7,0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′ independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the antisense strand is represented as formula (IIc), N_(b)′represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7,0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′ independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the antisense strand is represented as formula (IId), each N_(b)′independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides. Preferably, N_(b) is 0, 1, 2, 3, 4,5 or 6.

In other embodiments, k is 0 and 1 is 0 and the antisense strand may berepresented by the formula:

5′n _(p′)-N_(a′)-Y′Y′Y′-N_(a′)-n _(q′)3′  (Ia).

When the antisense strand is represented as formula (IIa), each N_(a)′independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

Each of X′, Y′ and Z′ may be the same or different from each other.

Each nucleotide of the sense strand and antisense strand may beindependently modified with LNA, HNA, CeNA, 2′-methoxyethyl,2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-hydroxyl, or 2′-fluoro. Forexample, each nucleotide of the sense strand and antisense strand isindependently modified with 2′-O-methyl or 2′-fluoro. Each X, Y, Z, X′,Y′ and Z′, in particular, may represent a 2′-O-methyl modification or a2′-fluoro modification.

In one embodiment, the sense strand of the RNAi agent may contain YYYmotif occurring at 9, 10 and 11 positions of the strand when the duplexregion is 21 nt, the count starting from the 1^(st) nucleotide from the5′-end, or optionally, the count starting at the 1^(st) pairednucleotide within the duplex region, from the 5′-end; and Y represents2′-F modification. The sense strand may additionally contain XXX motifor ZZZ motifs as wing modifications at the opposite end of the duplexregion; and XXX and ZZZ each independently represents a 2′-OMemodification or 2′-F modification.

In one embodiment the antisense strand may contain Y′Y′Y′ motifoccurring at positions 11, 12, 13 of the strand, the count starting fromthe 1^(st) nucleotide from the 5′-end, or optionally, the count startingat the 1^(st) paired nucleotide within the duplex region, from the5′-end; and Y′ represents 2′-O-methyl modification. The antisense strandmay additionally contain X′X′X′ motif or Z′Z′Z′ motifs as wingmodifications at the opposite end of the duplex region; and X′X′X′ andZ′Z′Z′ each independently represents a 2′-OMe modification or 2′-Fmodification.

The sense strand represented by any one of the above formulas (Ia),(Ib), (Ic), and (Id) forms a duplex with a antisense strand beingrepresented by any one of formulas (IIa), (IIb), (IIc), and (IId),respectively.

Accordingly, the RNAi agents for use in the methods of the invention maycomprise a sense strand and an antisense strand, each strand having 14to 30 nucleotides, the RNAi duplex represented by formula (III):

sense: 5′n _(p)-N_(a)-(XXX)_(i)-N_(b)-YYY-N_(b)-(ZZZ)_(j)-N_(a)-n _(q)3′

antisense: 3′n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)-(Z′Z′Z′)_(l)-N_(a)′-n_(q)′5′   (III)

wherein:

i, j, k, and l are each independently 0 or 1;

p, p′, q, and q′ are each independently 0-6;

each N_(a) and N_(a) independently represents an oligonucleotidesequence comprising 0-25 modified nucleotides, each sequence comprisingat least two differently modified nucleotides;

each N_(b) and N_(b) independently represents an oligonucleotidesequence comprising 0-10 modified nucleotides;

wherein

each n_(p)′, n_(p), n_(q)′, and n_(q), each of which may or may not bepresent, independently represents an overhang nucleotide; and

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides.

In one embodiment, i is 0 and j is 0; or i is 1 and j is 0; or i is 0and j is 1; or both i and j are 0; or both i and j are 1. In anotherembodiment, k is 0 and 1 is 0; or k is 1 and 1 is 0; k is 0 and 1 is 1;or both k and l are 0; or both k and l are 1.

Exemplary combinations of the sense strand and antisense strand forminga RNAi duplex include the formulas below:

5′n _(p)-N_(a)-YYY-N_(a)-n _(q)3′

3′n _(p)′-N_(a)′-Y′Y′Y′-N_(a) ′n _(q)′5′   (IIIa)

5′n _(p)-N_(a)-YYY-N_(b)-ZZZ-N_(a)-n _(q)3′

3′n _(p)′-N_(a)′-Y′Y′Y′-N_(b)′-Z′Z′Z′-N_(a) ′n _(q)′5′   (IIIb)

5′n _(p)-N_(a)-XXX-N_(b)-YYY-N_(a)-n _(q)3′

3′n _(p)′-N_(a)′-X′X′X′-N_(b)′-Y′Y′Y′-N_(a)′-n _(q)′5′   (IIIc)

5′n _(p)-N_(a)-XXX-N_(b)-YYY-N_(b)-ZZZ-N_(a)-n _(q)3′

3′n _(p)′-N_(a)′-X′X′X′-N_(b)′-Y′Y′Y′-N_(b)-Z′Z′Z′-N_(a)-n _(q)′5′  (IIId)

When the RNAi agent is represented by formula (IIIa), each N_(a)independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

When the RNAi agent is represented by formula (IIIb), each N_(b)independently represents an oligonucleotide sequence comprising 1-10,1-7, 1-5 or 1-4 modified nucleotides. Each N_(a) independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the RNAi agent is represented as formula (IIIc), each N_(b), N_(b)′independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

When the RNAi agent is represented as formula (IIId), each N_(b), N_(b)′independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a),N_(a)′ independently represents an oligonucleotide sequence comprising2-20, 2-15, or 2-10 modified nucleotides. Each of N_(a), N_(a)′, N_(b)and N_(b)′ independently comprises modifications of alternating pattern.

Each of X, Y and Z in formulas (III), (IIIa), (IIIb), (IIIc), and (IIId)may be the same or different from each other.

When the RNAi agent is represented by formula (III), (IIIa), (IIIb),(IIIc), and (IIId), at least one of the Y nucleotides may form a basepair with one of the Y′ nucleotides. Alternatively, at least two of theY nucleotides form base pairs with the corresponding Y′ nucleotides; orall three of the Y nucleotides all form base pairs with thecorresponding Y′ nucleotides.

When the RNAi agent is represented by formula (IIIb) or (IIId), at leastone of the Z nucleotides may form a base pair with one of the Z′nucleotides. Alternatively, at least two of the Z nucleotides form basepairs with the corresponding Z′ nucleotides; or all three of the Znucleotides all form base pairs with the corresponding Z′ nucleotides.

When the RNAi agent is represented as formula (IIIc) or (IIId), at leastone of the X nucleotides may form a base pair with one of the X′nucleotides. Alternatively, at least two of the X nucleotides form basepairs with the corresponding X′ nucleotides; or all three of the Xnucleotides all form base pairs with the corresponding X′ nucleotides.

In one embodiment, the modification on the Y nucleotide is differentthan the modification on the Y′ nucleotide, the modification on the Znucleotide is different than the modification on the Z′ nucleotide,and/or the modification on the X nucleotide is different than themodification on the X′ nucleotide.

In one embodiment, when the RNAi agent is represented by formula (IIId),the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications. Inanother embodiment, when the RNAi agent is represented by formula(IIId), the N_(a) modifications are 2′-O-methyl or 2′-fluoromodifications and n_(p)′>0 and at least one n_(p)′ is linked to aneighboring nucleotide a via phosphorothioate linkage. In yet anotherembodiment, when the RNAi agent is represented by formula (IIId), theN_(a) modifications are 2′-O-methyl or 2′-fluoro modifications, n_(p)′>0and at least one n_(p)′ is linked to a neighboring nucleotide viaphosphorothioate linkage, and the sense strand is conjugated to one ormore GalNAc derivatives attached through a bivalent or trivalentbranched linker. In another embodiment, when the RNAi agent isrepresented by formula (IIId), the N_(a) modifications are 2′-O-methylor 2′-fluoro modifications, n_(p)′>0 and at least one n_(p)′ is linkedto a neighboring nucleotide via phosphorothioate linkage, the sensestrand comprises at least one phosphorothioate linkage, and the sensestrand is conjugated to one or more GalNAc derivatives attached througha bivalent or trivalent branched linker.

In one embodiment, when the RNAi agent is represented by formula (IIIa),the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications,n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotidevia phosphorothioate linkage, the sense strand comprises at least onephosphorothioate linkage, and the sense strand is conjugated to one ormore GalNAc derivatives attached through a bivalent or trivalentbranched linker.

In one embodiment, the RNAi agent is a multimer containing at least twoduplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and(IIId), wherein the duplexes are connected by a linker. The linker canbe cleavable or non-cleavable. Optionally, the multimer furthercomprises a ligand. Each of the duplexes can target the same gene or twodifferent genes; or each of the duplexes can target same gene at twodifferent target sites.

In one embodiment, the RNAi agent is a multimer containing three, four,five, six or more duplexes represented by formula (III), (IIIa), (IIIb),(IIIc), and (IIId), wherein the duplexes are connected by a linker. Thelinker can be cleavable or non-cleavable. Optionally, the multimerfurther comprises a ligand. Each of the duplexes can target the samegene or two different genes; or each of the duplexes can target samegene at two different target sites.

In one embodiment, two RNAi agents represented by formula (III), (IIIa),(IIIb), (IIIc), and (IIId) are linked to each other at the 5′ end, andone or both of the 3′ ends and are optionally conjugated to to a ligand.Each of the agents can target the same gene or two different genes; oreach of the agents can target same gene at two different target sites.

Various publications describe multimeric RNAi agents that can be used inthe methods of the invention. Such publications include WO2007/091269,U.S. Pat. No. 7,858,769, WO2010/141511, WO2007/117686, WO2009/014887 andWO2011/031520 the entire contents of each of which are herebyincorporated herein by reference.

The RNAi agent that contains conjugations of one or more carbohydratemoieties to a RNAi agent can optimize one or more properties of the RNAiagent. In many cases, the carbohydrate moiety will be attached to amodified subunit of the RNAi agent. For example, the ribose sugar of oneor more ribonucleotide subunits of a dsRNA agent can be replaced withanother moiety, e.g., a non-carbohydrate (preferably cyclic) carrier towhich is attached a carbohydrate ligand. A ribonucleotide subunit inwhich the ribose sugar of the subunit has been so replaced is referredto herein as a ribose replacement modification subunit (RRMS). A cycliccarrier may be a carbocyclic ring system, i.e., all ring atoms arecarbon atoms, or a heterocyclic ring system, i.e., one or more ringatoms may be a heteroatom, e.g., nitrogen, oxygen, sulfur. The cycliccarrier may be a monocyclic ring system, or may contain two or morerings, e.g. fused rings. The cyclic carrier may be a fully saturatedring system, or it may contain one or more double bonds.

The ligand may be attached to the polynucleotide via a carrier. Thecarriers include (i) at least one “backbone attachment point,”preferably two “backbone attachment points” and (ii) at least one“tethering attachment point.” A “backbone attachment point” as usedherein refers to a functional group, e.g. a hydroxyl group, orgenerally, a bond available for, and that is suitable for incorporationof the carrier into the backbone, e.g., the phosphate, or modifiedphosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A“tethering attachment point” (TAP) in some embodiments refers to aconstituent ring atom of the cyclic carrier, e.g., a carbon atom or aheteroatom (distinct from an atom which provides a backbone attachmentpoint), that connects a selected moiety. The moiety can be, e.g., acarbohydrate, e.g. monosaccharide, disaccharide, trisaccharide,tetrasaccharide, oligosaccharide and polysaccharide. Optionally, theselected moiety is connected by an intervening tether to the cycliccarrier. Thus, the cyclic carrier will often include a functional group,e.g., an amino group, or generally, provide a bond, that is suitable forincorporation or tethering of another chemical entity, e.g., a ligand tothe constituent ring.

The RNAi agents may be conjugated to a ligand via a carrier, wherein thecarrier can be cyclic group or acyclic group; preferably, the cyclicgroup is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl,imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane,oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl,isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl and anddecalin; preferably, the acyclic group is selected from serinol backboneor diethanolamine backbone.

In certain specific embodiments, the RNAi agent for use in the methodsof the invention is an agent selected from the group of agents listed inany one of Tables 1, 2, 4, 5, 8, 10, and 12. In one embodiment, when theagent is an agent listed in Table 12, the agent may lack a terminal dT.

The present invention further includes double-stranded RNAi agentscomprising any one of the sequences listed in any one of Tables 1, 2, 4,5, 8, 10, and 12 which comprise a 5′ phosphate or phosphate mimetic onthe antisense strand (see, e.g., PCT Publication No. WO 2011005860).Further, the present invention includes double-stranded RNAi agentscomprising any one of the sequences listed in any one of Tables 1, 2, 4,5, 8, 10, and 12 which include a 2′fluoro group in place of a 2′-OMegroup at the 5′end of the sense strand.

These agents may further comprise a ligand.

In one embodiment, the agent is AD-60940 (sense strand:CfsusGfgUfaUfuUfCfCfuAfgGfgUfaCfaAfL96; antisense strand:usUfsgUfaCfcCfuAfggaAfaUfaCfcAfgsasg).

A. Ligands

The double-stranded RNA (dsRNA) agents of the invention may optionallybe conjugated to one or more ligands. The ligand can be attached to thesense strand, antisense strand or both strands, at the 3′-end, 5′-end orboth ends. For instance, the ligand may be conjugated to the sensestrand. In preferred embodiments, the ligand is conjugated to the 3′-endof the sense strand. In one preferred embodiment, the ligand is a GalNAcligand. In particularly preferred embodiments, the ligand is GalNAc₃:

In some embodiments, the ligand, e.g., GalNAc ligand, is attached to the3′ end of the RNAi agent. In one embodiment, the RNAi agent isconjugated to the ligand, e.g., GalNAc ligand, as shown in the followingschematic

wherein X is O or S. In one embodiment, X is O.

A wide variety of entities can be coupled to the RNAi agents of thepresent invention. Preferred moieties are ligands, which are coupled,preferably covalently, either directly or indirectly via an interveningtether.

In preferred embodiments, a ligand alters the distribution, targeting orlifetime of the molecule into which it is incorporated. In preferredembodiments a ligand provides an enhanced affinity for a selectedtarget, e.g., molecule, cell or cell type, compartment, receptor e.g., acellular or organ compartment, tissue, organ or region of the body, as,e.g., compared to a species absent such a ligand. Ligands providingenhanced affinity for a selected target are also termed targetingligands.

Some ligands can have endosomolytic properties. The endosomolyticligands promote the lysis of the endosome and/or transport of thecomposition of the invention, or its components, from the endosome tothe cytoplasm of the cell. The endosomolytic ligand may be a polyanionicpeptide or peptidomimetic which shows pH-dependent membrane activity andfusogenicity. In one embodiment, the endosomolytic ligand assumes itsactive conformation at endosomal pH. The “active” conformation is thatconformation in which the endosomolytic ligand promotes lysis of theendosome and/or transport of the composition of the invention, or itscomponents, from the endosome to the cytoplasm of the cell. Exemplaryendosomolytic ligands include the GALA peptide (Subbarao et al.,Biochemistry, 1987, 26: 2964-2972), the EALA peptide (Vogel et al., J.Am. Chem. Soc., 1996, 118: 1581-1586), and their derivatives (Turk etal., Biochem. Biophys. Acta, 2002, 1559: 56-68). In one embodiment, theendosomolytic component may contain a chemical group (e.g., an aminoacid) which will undergo a change in charge or protonation in responseto a change in pH. The endosomolytic component may be linear orbranched.

Ligands can improve transport, hybridization, and specificity propertiesand may also improve nuclease resistance of the resultant natural ormodified oligoribonucleotide, or a polymeric molecule comprising anycombination of monomers described herein and/or natural or modifiedribonucleotides.

Ligands in general can include therapeutic modifiers, e.g., forenhancing uptake; diagnostic compounds or reporter groups e.g., formonitoring distribution; cross-linking agents; and nuclease-resistanceconferring moieties. General examples include lipids, steroids,vitamins, sugars, proteins, peptides, polyamines, and peptide mimics.

Ligands can include a naturally occurring substance, such as a protein(e.g., human serum albumin (HSA), low-density lipoprotein (LDL),high-density lipoprotein (HDL), or globulin); a carbohydrate (e.g., adextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronicacid); or a lipid. The ligand may also be a recombinant or syntheticmolecule, such as a synthetic polymer, e.g., a synthetic polyamino acid,an oligonucleotide (e.g., an aptamer). Examples of polyamino acidsinclude polyamino acid is a polylysine (PLL), poly L-aspartic acid, polyL-glutamic acid, styrene-maleic acid anhydride copolymer,poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydridecopolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA),polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane,poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, orpolyphosphazine. Example of polyamines include: polyethylenimine,polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic lipid, cationic porphyrin,quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucosamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, biotin, an RGD peptide, an RGDpeptide mimetic or an aptamer.

Other examples of ligands include dyes, intercalating agents (e.g.,acridines), cross-linkers (e.g., psoralene, mitomycin C), porphyrins(TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases or a chelator(e.g., EDTA), lipophilic molecules, e.g., cholesterol, cholic acid,adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl,substituted alkyl, radiolabeled markers, enzymes, haptens (e.g.,biotin), transport/absorption facilitators (e.g., aspirin, vitamin E,folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole,histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a cancercell, endothelial cell, or bone cell. Ligands may also include hormonesand hormone receptors. They can also include non-peptidic species, suchas lipids, lectins, carbohydrates, vitamins, cofactors, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucosamine multivalent mannose, multivalent fucose, oraptamers. The ligand can be, for example, a lipopolysaccharide, anactivator of p38 MAP kinase, or an activator of NF-κB.

The ligand can be a substance, e.g., a drug, which can increase theuptake of the iRNA agent into the cell, for example, by disrupting thecell's cytoskeleton, e.g., by disrupting the cell's microtubules,microfilaments, and/or intermediate filaments. The drug can be, forexample, taxon, vincristine, vinblastine, cytochalasin, nocodazole,japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, ormyoservin.

The ligand can increase the uptake of the oligonucleotide into the cellby, for example, activating an inflammatory response. Exemplary ligandsthat would have such an effect include tumor necrosis factor alpha(TNFalpha), interleukin-1 beta, or gamma interferon.

In one aspect, the ligand is a lipid or lipid-based molecule. Such alipid or lipid-based molecule preferably binds a serum protein, e.g.,human serum albumin (HSA). An HSA binding ligand allows for distributionof the conjugate to a target tissue, e.g., a non-kidney target tissue ofthe body. For example, the target tissue can be the liver, includingparenchymal cells of the liver. Other molecules that can bind HSA canalso be used as ligands. For example, naproxen or aspirin can be used. Alipid or lipid-based ligand can (a) increase resistance to degradationof the conjugate, (b) increase targeting or transport into a target cellor cell membrane, and/or (c) can be used to adjust binding to a serumprotein, e.g., HSA.

A lipid based ligand can be used to modulate, e.g., control the bindingof the conjugate to a target tissue. For example, a lipid or lipid-basedligand that binds to HSA more strongly will be less likely to betargeted to the kidney and therefore less likely to be cleared from thebody. A lipid or lipid-based ligand that binds to HSA less strongly canbe used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably,it binds HSA with a sufficient affinity such that the conjugate will bepreferably distributed to a non-kidney tissue. However, it is preferredthat the affinity not be so strong that the HSA-ligand binding cannot bereversed.

In another preferred embodiment, the lipid based ligand binds HSA weaklyor not at all, such that the conjugate will be preferably distributed tothe kidney. Other moieties that target to kidney cells can also be usedin place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which istaken up by a target cell, e.g., a proliferating cell. These areparticularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells. Exemplary vitamins include vitamin A, E, and K. Otherexemplary vitamins include B vitamins, e.g., folic acid, B12,riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up bycancer cells. Also included are HAS, low density lipoprotein (LDL) andhigh-density lipoprotein (HDL).

In another aspect, the ligand is a cell-permeation agent, preferably ahelical cell-permeation agent. Preferably, the agent is amphipathic. Anexemplary agent is a peptide such as tat or antennopedia. If the agentis a peptide, it can be modified, including a peptidylmimetic,invertomers, non-peptide or pseudo-peptide linkages, and use of D-aminoacids. The helical agent is preferably an alpha-helical agent, whichpreferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (alsoreferred to herein as an oligopeptidomimetic) is a molecule capable offolding into a defined three-dimensional structure similar to a naturalpeptide. The peptide or peptidomimetic moiety can be about 5-50 aminoacids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 aminoacids long.

A peptide or peptidomimetic can be, for example, a cell permeationpeptide, cationic peptide, amphipathic peptide, or hydrophobic peptide(e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety canbe a dendrimer peptide, constrained peptide or crosslinked peptide. Inanother alternative, the peptide moiety can include a hydrophobicmembrane translocation sequence (MTS). An exemplary hydrophobicMTS-containing peptide is RFGF having the amino acid sequenceAAVALLPAVLLALLAP (SEQ ID NO:11). An RFGF analogue (e.g., amino acidsequence AALLPVLLAAP (SEQ ID NO:12)) containing a hydrophobic MTS canalso be a targeting moiety. The peptide moiety can be a “delivery”peptide, which can carry large polar molecules including peptides,oligonucleotides, and protein across cell membranes. For example,sequences from the HIV Tat protein (GRKKRRQRRRPPQ) (SEQ ID NO:13) andthe Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK) (SEQ ID NO:14)have been found to be capable of functioning as delivery peptides. Apeptide or peptidomimetic can be encoded by a random sequence of DNA,such as a peptide identified from a phage-display library, orone-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature,354:82-84, 1991). Preferably the peptide or peptidomimetic tethered toan iRNA agent via an incorporated monomer unit is a cell targetingpeptide such as an arginine-glycine-aspartic acid (RGD)-peptide, or RGDmimic. A peptide moiety can range in length from about 5 amino acids toabout 40 amino acids. The peptide moieties can have a structuralmodification, such as to increase stability or direct conformationalproperties. Any of the structural modifications described below can beutilized. An RGD peptide moiety can be used to target a tumor cell, suchas an endothelial tumor cell or a breast cancer tumor cell (Zitzmann etal., Cancer Res., 62:5139-43, 2002). An RGD peptide can facilitatetargeting of an iRNA agent to tumors of a variety of other tissues,including the lung, kidney, spleen, or liver (Aoki et al., Cancer GeneTherapy 8:783-787, 2001). Preferably, the RGD peptide will facilitatetargeting of an iRNA agent to the kidney. The RGD peptide can be linearor cyclic, and can be modified, e.g., glycosylated or methylated tofacilitate targeting to specific tissues. For example, a glycosylatedRGD peptide can deliver an iRNA agent to a tumor cell expressing α_(ν)β₃(Haubner et al., Jour. Nucl. Med., 42:326-336, 2001). Peptides thattarget markers enriched in proliferating cells can be used. For example,RGD containing peptides and peptidomimetics can target cancer cells, inparticular cells that exhibit an integrin. Thus, one could use RGDpeptides, cyclic peptides containing RGD, RGD peptides that includeD-amino acids, as well as synthetic RGD mimics. In addition to RGD, onecan use other moieties that target the integrin ligand. Generally, suchligands can be used to control proliferating cells and angiogeneis.Preferred conjugates of this type of ligand target PECAM-1, VEGF, orother cancer gene, e.g., a cancer gene described herein.

A “cell permeation peptide” is capable of permeating a cell, e.g., amicrobial cell, such as a bacterial or fungal cell, or a mammalian cell,such as a human cell. A microbial cell-permeating peptide can be, forexample, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), adisulfide bond-containing peptide (e.g., α-defensin, β-defensin orbactenecin), or a peptide containing only one or two dominating aminoacids (e.g., PR-39 or indolicidin). A cell permeation peptide can alsoinclude a nuclear localization signal (NLS). For example, a cellpermeation peptide can be a bipartite amphipathic peptide, such as MPG,which is derived from the fusion peptide domain of HIV-1 gp41 and theNLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.31:2717-2724, 2003).

In one embodiment, a targeting peptide can be an amphipathic α-helicalpeptide. Exemplary amphipathic α-helical peptides include, but are notlimited to, cecropins, lycotoxins, paradaxins, buforin, CPF,bombinin-like peptide (BLP), cathelicidins, ceratotoxins, S. clavapeptides, hagfish intestinal antimicrobial peptides (HFIAPs),magainines, brevinins-2, dermaseptins, melittins, pleurocidin, H₂Apeptides, Xenopus peptides, esculentinis-1, and caerins. A number offactors will preferably be considered to maintain the integrity of helixstability. For example, a maximum number of helix stabilization residueswill be utilized (e.g., leu, ala, or lys), and a minimum number helixdestabilization residues will be utilized (e.g., proline, or cyclicmonomeric units. The capping residue will be considered (for example Glyis an exemplary N-capping residue and/or C-terminal amidation can beused to provide an extra H-bond to stabilize the helix. Formation ofsalt bridges between residues with opposite charges, separated by i±3,or i±4 positions can provide stability. For example, cationic residuessuch as lysine, arginine, homo-arginine, ornithine or histidine can formsalt bridges with the anionic residues glutamate or aspartate.

Peptide and peptidomimetic ligands include those having naturallyoccurring or modified peptides, e.g., D or L peptides; α, β, or γpeptides; N-methyl peptides; azapeptides; peptides having one or moreamide, i.e., peptide, linkages replaced with one or more urea, thiourea,carbamate, or sulfonyl urea linkages; or cyclic peptides.

The targeting ligand can be any ligand that is capable of targeting aspecific receptor. Examples are: folate, GalNAc, galactose, mannose,mannose-6P, clusters of sugars such as GalNAc cluster, mannose cluster,galactose cluster, or an apatamer. A cluster is a combination of two ormore sugar units. The targeting ligands also include integrin receptorligands, Chemokine receptor ligands, transferrin, biotin, serotoninreceptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL and HDLligands. The ligands can also be based on nucleic acid, e.g., anaptamer. The aptamer can be unmodified or have any combination ofmodifications disclosed herein.

Endosomal release agents include imidazoles, poly or oligoimidazoles,PEIs, peptides, fusogenic peptides, polycaboxylates, polyacations,masked oligo or poly cations or anions, acetals, polyacetals,ketals/polyketyals, orthoesters, polymers with masked or unmaskedcationic or anionic charges, dendrimers with masked or unmasked cationicor anionic charges.

PK modulator stands for pharmacokinetic modulator. PK modulators includelipophiles, bile acids, steroids, phospholipid analogues, peptides,protein binding agents, PEG, vitamins etc. Exemplary PK modulatorsinclude, but are not limited to, cholesterol, fatty acids, cholic acid,lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids,sphingolipids, naproxen, ibuprofen, vitamin E, biotin etc.Oligonucleotides that comprise a number of phosphorothioate linkages arealso known to bind to serum protein, thus short oligonucleotides, e.g.,oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases,comprising multiple phosphorothioate linkages in the backbone are alsoamenable to the present invention as ligands (e.g., as PK modulatingligands).

In addition, aptamers that bind serum components (e.g., serum proteins)are also amenable to the present invention as PK modulating ligands.

Other ligand conjugates amenable to the invention are described in U.S.patent application Ser. No. 10/916,185, filed Aug. 10, 2004; U.S. Ser.No. 10/946,873, filed Sep. 21, 2004; U.S. Ser. No. 10/833,934, filedAug. 3, 2007; U.S. Ser. No. 11/115,989 filed Apr. 27, 2005 and U.S. Ser.No. 11/944,227 filed Nov. 21, 2007, which are incorporated by referencein their entireties for all purposes.

When two or more ligands are present, the ligands can all have sameproperties, all have different properties or some ligands have the sameproperties while others have different properties. For example, a ligandcan have targeting properties, have endosomolytic activity or have PKmodulating properties. In a preferred embodiment, all the ligands havedifferent properties.

Ligands can be coupled to the oligonucleotides at various places, forexample, 3′-end, 5′-end, and/or at an internal position. In preferredembodiments, the ligand is attached to the oligonucleotides via anintervening tether, e.g., a carrier described herein. The ligand ortethered ligand may be present on a monomer when the monomer isincorporated into the growing strand. In some embodiments, the ligandmay be incorporated via coupling to a “precursor” monomer after the“precursor” monomer has been incorporated into the growing strand. Forexample, a monomer having, e.g., an amino-terminated tether (i.e.,having no associated ligand), e.g., TAP-(CH₂)_(n)NH₂ may be incorporatedinto a growing oligonucleotide strand. In a subsequent operation, i.e.,after incorporation of the precursor monomer into the strand, a ligandhaving an electrophilic group, e.g., a pentafluorophenyl ester oraldehyde group, can subsequently be attached to the precursor monomer bycoupling the electrophilic group of the ligand with the terminalnucleophilic group of the precursor monomer's tether.

In another example, a monomer having a chemical group suitable fortaking part in Click Chemistry reaction may be incorporated, e.g., anazide or alkyne terminated tether/linker. In a subsequent operation,i.e., after incorporation of the precursor monomer into the strand, aligand having complementary chemical group, e.g. an alkyne or azide canbe attached to the precursor monomer by coupling the alkyne and theazide together.

For double-stranded oligonucleotides, ligands can be attached to one orboth strands. In some embodiments, a double-stranded iRNA agent containsa ligand conjugated to the sense strand. In other embodiments, adouble-stranded iRNA agent contains a ligand conjugated to the antisensestrand.

In some embodiments, ligand can be conjugated to nucleobases, sugarmoieties, or internucleosidic linkages of nucleic acid molecules.Conjugation to purine nucleobases or derivatives thereof can occur atany position including, endocyclic and exocyclic atoms. In someembodiments, the 2-, 6-, 7-, or 8-positions of a purine nucleobase areattached to a conjugate moiety. Conjugation to pyrimidine nucleobases orderivatives thereof can also occur at any position. In some embodiments,the 2-, 5-, and 6-positions of a pyrimidine nucleobase can besubstituted with a conjugate moiety. Conjugation to sugar moieties ofnucleosides can occur at any carbon atom. Example carbon atoms of asugar moiety that can be attached to a conjugate moiety include the 2′,3′, and 5′ carbon atoms. The 1′ position can also be attached to aconjugate moiety, such as in an abasic residue. Internucleosidiclinkages can also bear conjugate moieties. For phosphorus-containinglinkages (e.g., phosphodiester, phosphorothioate, phosphorodithiotate,phosphoroamidate, and the like), the conjugate moiety can be attacheddirectly to the phosphorus atom or to an O, N, or S atom bound to thephosphorus atom. For amine- or amide-containing internucleosidiclinkages (e.g., PNA), the conjugate moiety can be attached to thenitrogen atom of the amine or amide or to an adjacent carbon atom.

Any suitable ligand in the field of RNA interference may be used,although the ligand is typically a carbohydrate e.g. monosaccharide(such as GalNAc), disaccharide, trisaccharide, tetrasaccharide,polysaccharide.

Linkers that conjugate the ligand to the nucleic acid include thosediscussed above. For example, the ligand can be one or more GalNAc(N-acetylglucosamine) derivatives attached through a bivalent ortrivalent branched linker.

In one embodiment, the dsRNA of the invention is conjugated to abivalent and trivalent branched linkers include the structures shown inany of formula (IV)-(VII):

wherein:

q^(2A), q^(2B), q^(3A), q^(3B), q4^(A), q^(4B), q^(5A), q^(5B) andq^(5C) represent independently for each occurrence 0-20 and wherein therepeating unit can be the same or different;

P^(2A), P^(2B), P^(3A), P^(3B), P^(4A), P^(4B), P^(5A), P^(5B), P^(5C),T^(2A), T^(2B), T^(3A), T^(3B), T^(4A), T^(4B), T^(4A), T^(5B), T^(5C)are each independently for each occurrence absent, CO, NH, O, S, OC(O),NHC(O), CH₂, CH₂NH or CH₂O;

Q^(2A), Q^(2B), Q^(3A), Q^(3B), Q^(4A), Q^(4B), Q^(5A), Q^(5B), Q^(5C)are independently for each occurrence absent, alkylene, substitutedalkylene wherein one or more methylenes can be interrupted or terminatedby one or more of O, S, S(O), SO₂, N(R^(N)), C(R′)═C(R″), C≡C or C(O);

R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B), R^(5A), R^(5B), R^(5c)are each independently for each occurrence absent, NH, O, S, CH₂, C(O)O,C(O)NH, NHCH(R^(a))C(O), —C(O)—CH(R^(a))—NH—, CO, CH═N—O,

or heterocyclyl;

L^(2A), L^(2B), L^(3A), L^(3B), L^(4A), L^(4B), L^(5A), L^(5B) andL^(5C) represent the ligand; i.e. each independently for each occurrencea monosaccharide (such as GalNAc), disaccharide, trisaccharide,tetrasaccharide, oligosaccharide, or polysaccharide; and

R^(a) is H or amino acid side chain.

Trivalent conjugating GalNAc derivatives are particularly useful for usewith RNAi agents for inhibiting the expression of a target gene, such asthose of formula (VII):

wherein L^(5A), L^(5B) and L^(5C) represent a monosaccharide, such asGalNAc derivative. Examples of suitable bivalent and trivalent branchedlinker groups conjugating GalNAc derivatives include, but are notlimited to, the following compounds:

In other embodiments, the RNAi agent for use in the methods of theinvention is AD-59743.

III. Delivery of an iRNA of the Invention

The delivery of an iRNA agent of the invention to a cell e.g., a cellwithin a subject, such as a human subject (e.g., a subject in needthereof, such as a subject having a TMPRSS6 associated disorder, such asa hemochromatosis) can be achieved in a number of different ways. Forexample, delivery may be performed by contacting a cell with an iRNA ofthe invention either in vitro or in vivo. In vivo delivery may also beperformed directly by administering a composition comprising an iRNA,e.g., a dsRNA, to a subject. Alternatively, in vivo delivery may beperformed indirectly by administering one or more vectors that encodeand direct the expression of the iRNA. These alternatives are discussedfurther below.

In general, any method of delivering a nucleic acid molecule (in vitroor in vivo) can be adapted for use with an iRNA of the invention (seee.g., Akhtar S. and Julian R L. (1992) Trends Cell. Biol. 2(5):139-144and WO94/02595, which are incorporated herein by reference in theirentireties). For in vivo delivery, factors to consider in order todeliver an iRNA molecule include, for example, biological stability ofthe delivered molecule, prevention of non-specific effects, andaccumulation of the delivered molecule in the target tissue. Thenon-specific effects of an iRNA can be minimized by localadministration, for example, by direct injection or implantation into atissue or topically administering the preparation. Local administrationto a treatment site maximizes local concentration of the agent, limitsthe exposure of the agent to systemic tissues that can otherwise beharmed by the agent or that can degrade the agent, and permits a lowertotal dose of the iRNA molecule to be administered. Several studies haveshown successful knockdown of gene products when an iRNA is administeredlocally. For example, intraocular delivery of a VEGF dsRNA byintravitreal injection in cynomolgus monkeys (Tolentino, M J., et al(2004) Retina 24:132-138) and subretinal injections in mice (Reich, SJ., et al (2003) Mol. Vis. 9:210-216) were both shown to preventneovascularization in an experimental model of age-related maculardegeneration. In addition, direct intratumoral injection of a dsRNA inmice reduces tumor volume (Pille, J., et al (2005) Mol. Ther.11:267-274) and can prolong survival of tumor-bearing mice (Kim, W J.,et al (2006) Mol. Ther. 14:343-350; Li, S., et al (2007) Mol. Ther.15:515-523). RNA interference has also shown success with local deliveryto the CNS by direct injection (Dorn, G., et al. (2004) Nucleic Acids32:e49; Tan, P H., et al (2005) Gene Ther. 12:59-66; Makimura, H., et al(2002) BMC Neurosci. 3:18; Shishkina, G T., et al (2004) Neuroscience129:521-528; Thakker, E R., et al (2004) Proc. Natl. Acad. Sci. U.S.A.101:17270-17275; Akaneya, Y., et al (2005) J. Neurophysiol. 93:594-602)and to the lungs by intranasal administration (Howard, K A., et al(2006) Mol. Ther. 14:476-484; Zhang, X., et al (2004) J. Biol. Chem.279:10677-10684; Bitko, V., et al (2005) Nat. Med. 11:50-55). Foradministering an iRNA systemically for the treatment of a disease, theRNA can be modified or alternatively delivered using a drug deliverysystem; both methods act to prevent the rapid degradation of the dsRNAby endo- and exo-nucleases in vivo. Modification of the RNA or thepharmaceutical carrier can also permit targeting of the iRNA compositionto the target tissue and avoid undesirable off-target effects. iRNAmolecules can be modified by chemical conjugation to lipophilic groupssuch as cholesterol to enhance cellular uptake and prevent degradation.For example, an iRNA directed against ApoB conjugated to a lipophiliccholesterol moiety was injected systemically into mice and resulted inknockdown of apoB mRNA in both the liver and jejunum (Soutschek, J., etal (2004) Nature 432:173-178). Conjugation of an iRNA to an aptamer hasbeen shown to inhibit tumor growth and mediate tumor regression in amouse model of prostate cancer (McNamara, J O., et al (2006) Nat.Biotechnol. 24:1005-1015). In an alternative embodiment, the iRNA can bedelivered using drug delivery systems such as a nanoparticle, adendrimer, a polymer, liposomes, or a cationic delivery system.Positively charged cationic delivery systems facilitate binding of aniRNA molecule (negatively charged) and also enhance interactions at thenegatively charged cell membrane to permit efficient uptake of an iRNAby the cell. Cationic lipids, dendrimers, or polymers can either bebound to an iRNA, or induced to form a vesicle or micelle (see e.g., KimS H., et al (2008) Journal of Controlled Release 129(2):107-116) thatencases an iRNA. The formation of vesicles or micelles further preventsdegradation of the iRNA when administered systemically. Methods formaking and administering cationic-iRNA complexes are well within theabilities of one skilled in the art (see e.g., Sorensen, D R., et al(2003) J. Mol. Biol 327:761-766; Verma, U N., et al (2003) Clin. CancerRes. 9:1291-1300; Arnold, A S et al (2007) J. Hypertens. 25:197-205,which are incorporated herein by reference in their entirety). Somenon-limiting examples of drug delivery systems useful for systemicdelivery of iRNAs include DOTAP (Sorensen, D R., et al (2003), supra;Verma, U N., et al (2003), supra), Oligofectamine, “solid nucleic acidlipid particles” (Zimmermann, T S., et al (2006) Nature 441:111-114),cardiolipin (Chien, P Y., et al (2005) Cancer Gene Ther. 12:321-328;Pal, A., et al (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine(Bonnet M E., et al (2008) Pharm. Res. August 16 Epub ahead of print;Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD)peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines(Tomalia, D A., et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., etal (1999) Pharm. Res. 16:1799-1804). In some embodiments, an iRNA formsa complex with cyclodextrin for systemic administration. Methods foradministration and pharmaceutical compositions of iRNAs andcyclodextrins can be found in U.S. Pat. No. 7,427,605, which is hereinincorporated by reference in its entirety.

A. Vector Encoded iRNAs of the Invention

iRNA targeting the TMPRSS6 gene can be expressed from transcriptionunits inserted into DNA or RNA vectors (see, e.g., Couture, A, et al.,TIG. (1996), 12:5-10; Skillern, A., et al., International PCTPublication No. WO 00/22113, Conrad, International PCT Publication No.WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). Expression can betransient (on the order of hours to weeks) or sustained (weeks to monthsor longer), depending upon the specific construct used and the targettissue or cell type. These transgenes can be introduced as a linearconstruct, a circular plasmid, or a viral vector, which can be anintegrating or non-integrating vector. The transgene can also beconstructed to permit it to be inherited as an extrachromosomal plasmid(Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

The individual strand or strands of an iRNA can be transcribed from apromoter on an expression vector. Where two separate strands are to beexpressed to generate, for example, a dsRNA, two separate expressionvectors can be co-introduced (e.g., by transfection or infection) into atarget cell. Alternatively each individual strand of a dsRNA can betranscribed by promoters both of which are located on the sameexpression plasmid. In one embodiment, a dsRNA is expressed as invertedrepeat polynucleotides joined by a linker polynucleotide sequence suchthat the dsRNA has a stem and loop structure.

iRNA expression vectors are generally DNA plasmids or viral vectors.Expression vectors compatible with eukaryotic cells, preferably thosecompatible with vertebrate cells, can be used to produce recombinantconstructs for the expression of an iRNA as described herein. Eukaryoticcell expression vectors are well known in the art and are available froma number of commercial sources. Typically, such vectors are providedcontaining convenient restriction sites for insertion of the desirednucleic acid segment. Delivery of iRNA expressing vectors can besystemic, such as by intravenous or intramuscular administration, byadministration to target cells ex-planted from the patient followed byreintroduction into the patient, or by any other means that allows forintroduction into a desired target cell.

iRNA expression plasmids can be transfected into target cells as acomplex with cationic lipid carriers (e.g., Oligofectamine) ornon-cationic lipid-based carriers (e.g., Transit-TKO™). Multiple lipidtransfections for iRNA-mediated knockdowns targeting different regionsof a target RNA over a period of a week or more are also contemplated bythe invention. Successful introduction of vectors into host cells can bemonitored using various known methods. For example, transienttransfection can be signaled with a reporter, such as a fluorescentmarker, such as Green Fluorescent Protein (GFP). Stable transfection ofcells ex vivo can be ensured using markers that provide the transfectedcell with resistance to specific environmental factors (e.g.,antibiotics and drugs), such as hygromycin B resistance.

Viral vector systems which can be utilized with the methods andcompositions described herein include, but are not limited to, (a)adenovirus vectors; (b) retrovirus vectors, including but not limited tolentiviral vectors, moloney murine leukemia virus, etc.; (c)adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h)picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g.,vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) ahelper-dependent or gutless adenovirus. Replication-defective virusescan also be advantageous. Different vectors will or will not becomeincorporated into the cells' genome. The constructs can include viralsequences for transfection, if desired. Alternatively, the construct canbe incorporated into vectors capable of episomal replication, e.g. EPVand EBV vectors. Constructs for the recombinant expression of an iRNAwill generally require regulatory elements, e.g., promoters, enhancers,etc., to ensure the expression of the iRNA in target cells. Otheraspects to consider for vectors and constructs are further describedbelow.

Vectors useful for the delivery of an iRNA will include regulatoryelements (promoter, enhancer, etc.) sufficient for expression of theiRNA in the desired target cell or tissue. The regulatory elements canbe chosen to provide either constitutive or regulated/inducibleexpression.

Expression of the iRNA can be precisely regulated, for example, by usingan inducible regulatory sequence that is sensitive to certainphysiological regulators, e.g., circulating glucose levels, or hormones(Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expressionsystems, suitable for the control of dsRNA expression in cells or inmammals include, for example, regulation by ecdysone, by estrogen,progesterone, tetracycline, chemical inducers of dimerization, andisopropyl-beta-D1-thiogalactopyranoside (IPTG). A person skilled in theart would be able to choose the appropriate regulatory/promoter sequencebased on the intended use of the iRNA transgene.

Viral vectors that contain nucleic acid sequences encoding an iRNA canbe used. For example, a retroviral vector can be used (see Miller etal., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectorscontain the components necessary for the correct packaging of the viralgenome and integration into the host cell DNA. The nucleic acidsequences encoding an iRNA are cloned into one or more vectors, whichfacilitate delivery of the nucleic acid into a patient. More detailabout retroviral vectors can be found, for example, in Boesen et al.,Biotherapy 6:291-302 (1994), which describes the use of a retroviralvector to deliver the mdr1 gene to hematopoietic stem cells in order tomake the stem cells more resistant to chemotherapy. Other referencesillustrating the use of retroviral vectors in gene therapy are: Cloweset al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141(1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel.3:110-114 (1993). Lentiviral vectors contemplated for use include, forexample, the HIV based vectors described in U.S. Pat. Nos. 6,143,520;5,665,557; and 5,981,276, which are herein incorporated by reference.

Adenoviruses are also contemplated for use in delivery of iRNAs of theinvention. Adenoviruses are especially attractive vehicles, e.g., fordelivering genes to respiratory epithelia. Adenoviruses naturally infectrespiratory epithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, CurrentOpinion in Genetics and Development 3:499-503 (1993) present a review ofadenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10(1994) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al.,Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992);Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT PublicationWO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). A suitableAV vector for expressing an iRNA featured in the invention, a method forconstructing the recombinant AV vector, and a method for delivering thevector into target cells, are described in Xia H et al. (2002), Nat.Biotech. 20: 1006-1010.

Adeno-associated virus (AAV) vectors may also be used to delivery aniRNA of the invention (Walsh et al., Proc. Soc. Exp. Biol. Med.204:289-300 (1993); U.S. Pat. No. 5,436,146). In one embodiment, theiRNA can be expressed as two separate, complementary single-stranded RNAmolecules from a recombinant AAV vector having, for example, either theU6 or H1 RNA promoters, or the cytomegalovirus (CMV) promoter. SuitableAAV vectors for expressing the dsRNA featured in the invention, methodsfor constructing the recombinant AV vector, and methods for deliveringthe vectors into target cells are described in Samulski R et al. (1987),J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70:520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat.Nos. 5,252,479; 5,139,941; International Patent Application No. WO94/13788; and International Patent Application No. WO 93/24641, theentire disclosures of which are herein incorporated by reference.

Another viral vector suitable for delivery of an iRNA of the inventionis a pox virus such as a vaccinia virus, for example an attenuatedvaccinia such as Modified Virus Ankara (MVA) or NYVAC, an avipox such asfowl pox or canary pox.

The tropism of viral vectors can be modified by pseudotyping the vectorswith envelope proteins or other surface antigens from other viruses, orby substituting different viral capsid proteins, as appropriate. Forexample, lentiviral vectors can be pseudotyped with surface proteinsfrom vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and thelike. AAV vectors can be made to target different cells by engineeringthe vectors to express different capsid protein serotypes; see, e.g.,Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosureof which is herein incorporated by reference.

The pharmaceutical preparation of a vector can include the vector in anacceptable diluent, or can include a slow release matrix in which thegene delivery vehicle is imbedded. Alternatively, where the completegene delivery vector can be produced intact from recombinant cells,e.g., retroviral vectors, the pharmaceutical preparation can include oneor more cells which produce the gene delivery system.

IV. Pharmaceutical Compositions of the Invention

The present invention also includes pharmaceutical compositions andformulations which include the iRNAs of the invention. In oneembodiment, provided herein are pharmaceutical compositions containingan iRNA, as described herein, and a pharmaceutically acceptable carrier.The pharmaceutical compositions containing the iRNA are useful fortreating a TMPRSS6 associated disease or disorder, e.g. hemochromatosis.Such pharmaceutical compositions are formulated based on the mode ofdelivery. One example is compositions that are formulated for systemicadministration via parenteral delivery, e.g., by intravenous (IV)delivery. Another example is compositions that are formulated for directdelivery into the brain parenchyma, e.g., by infusion into the brain,such as by continuous pump infusion.

The pharmaceutical compositions comprising RNAi agents of the inventionmay be, for example, solutions with or without a buffer, or compositionscontaining pharmaceutically acceptable carriers. Such compositionsinclude, for example, aqueous or crystalline compositions, liposomalformulations, micellar formulations, emulsions, and gene therapyvectors.

In the methods of the invention, the RNAi agent may be administered in asolution. A free RNAi agent may be administered in an unbufferedsolution, e.g., in saline or in water. Alternatively, the free siRNA mayalso be administered in a suitable buffer solution. The buffer solutionmay comprise acetate, citrate, prolamine, carbonate, or phosphate, orany combination thereof. In a preferred embodiment, the buffer solutionis phosphate buffered saline (PBS). The pH and osmolarity of the buffersolution containing the RNAi agent can be adjusted such that it issuitable for administering to a subject.

In some embodiments, the buffer solution further comprises an agent forcontrolling the osmolarity of the solution, such that the osmolarity iskept at a desired value, e.g., at the physiologic values of the humanplasma. Solutes which can be added to the buffer solution to control theosmolarity include, but are not limited to, proteins, peptides, aminoacids, non-metabolized polymers, vitamins, ions, sugars, metabolites,organic acids, lipids, or salts. In some embodiments, the agent forcontrolling the osmolarity of the solution is a salt. In certainembodiments, the agent for controlling the osmolarity of the solution issodium chloride or potassium chloride.

The pharmaceutical compositions of the invention may be administered indosages sufficient to inhibit expression of a TMPRSS6 gene.

In general, a suitable dose of an iRNA of the invention will be in therange of about 0.001 to about 200.0 milligrams per kilogram body weightof the recipient per day, generally in the range of about 1 to 50 mg perkilogram body weight per day. For example, the dsRNA can be administeredat about 0.01 mg/kg, about 0.05 mg/kg, about 0.5 mg/kg, about 1 mg/kg,about 1.5 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg about10 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, or about 50mg/kg per single dose.

For example, the RNAi agent, e.g., dsRNA, may be administered at a doseof about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3,1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3,4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8,5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3,7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8,8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg/kg.Values and ranges intermediate to the recited values are also intendedto be part of this invention.

In another embodiment, the RNAi agent, e.g., dsRNA, is administered at adose of about 0.1 to about 50 mg/kg, about 0.25 to about 50 mg/kg, about0.5 to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50mg/mg, about 1.5 to about 50 mg/kb, about 2 to about 50 mg/kg, about 2.5to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50mg/kg, about 15 to about 50 mg/kg, about 20 to about 50 mg/kg, about 20to about 50 mg/kg, about 25 to about 50 mg/kg, about 25 to about 50mg/kg, about 30 to about 50 mg/kg, about 35 to about 50 mg/kg, about 40to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.1 to about 45mg/kg, about 0.25 to about 45 mg/kg, about 0.5 to about 45 mg/kg, about0.75 to about 45 mg/kg, about 1 to about 45 mg/mg, about 1.5 to about 45mg/kb, about 2 to about 45 mg/kg, about 2.5 to about 45 mg/kg, about 3to about 45 mg/kg, about 3.5 to about 45 mg/kg, about 4 to about 45mg/kg, about 4.5 to about 45 mg/kg, about 5 to about 45 mg/kg, about 7.5to about 45 mg/kg, about 10 to about 45 mg/kg, about 15 to about 45mg/kg, about 20 to about 45 mg/kg, about 20 to about 45 mg/kg, about 25to about 45 mg/kg, about 25 to about 45 mg/kg, about 30 to about 45mg/kg, about 35 to about 45 mg/kg, about 40 to about 45 mg/kg, about 0.1to about 40 mg/kg, about 0.25 to about 40 mg/kg, about 0.5 to about 40mg/kg, about 0.75 to about 40 mg/kg, about 1 to about 40 mg/mg, about1.5 to about 40 mg/kb, about 2 to about 40 mg/kg, about 2.5 to about 40mg/kg, about 3 to about 40 mg/kg, about 3.5 to about 40 mg/kg, about 4to about 40 mg/kg, about 4.5 to about 40 mg/kg, about 5 to about 40mg/kg, about 7.5 to about 40 mg/kg, about 10 to about 40 mg/kg, about 15to about 40 mg/kg, about 20 to about 40 mg/kg, about 20 to about 40mg/kg, about 25 to about 40 mg/kg, about 25 to about 40 mg/kg, about 30to about 40 mg/kg, about 35 to about 40 mg/kg, about 0.1 to about 30mg/kg, about 0.25 to about 30 mg/kg, about 0.5 to about 30 mg/kg, about0.75 to about 30 mg/kg, about 1 to about 30 mg/mg, about 1.5 to about 30mg/kb, about 2 to about 30 mg/kg, about 2.5 to about 30 mg/kg, about 3to about 30 mg/kg, about 3.5 to about 30 mg/kg, about 4 to about 30mg/kg, about 4.5 to about 30 mg/kg, about 5 to about 30 mg/kg, about 7.5to about 30 mg/kg, about 10 to about 30 mg/kg, about 15 to about 30mg/kg, about 20 to about 30 mg/kg, about 20 to about 30 mg/kg, about 25to about 30 mg/kg, about 0.1 to about 20 mg/kg, about 0.25 to about 20mg/kg, about 0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about1 to about 20 mg/mg, about 1.5 to about 20 mg/kb, about 2 to about 20mg/kg, about 2.5 to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5to about 20 mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20mg/kg, about 5 to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10to about 20 mg/kg, or about 15 to about 20 mg/kg. Values and rangesintermediate to the recited values are also intended to be part of thisinvention.

For example, the RNAi agent, e.g., dsRNA, may be administered at a doseof about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2,3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2,6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7,7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2,9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg/kg. Values and rangesintermediate to the recited values are also intended to be part of thisinvention.

In another embodiment, the RNAi agent, e.g., dsRNA, is administered at adose of about 0.5 to about 50 mg/kg, about 0.75 to about 50 mg/kg, about1 to about 50 mg/mg, about 1.5 to about 50 mg/kg, about 2 to about 50mg/kg, about 2.5 to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5to about 50 mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50mg/kg, about 5 to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10to about 50 mg/kg, about 15 to about 50 mg/kg, about 20 to about 50mg/kg, about 20 to about 50 mg/kg, about 25 to about 50 mg/kg, about 25to about 50 mg/kg, about 30 to about 50 mg/kg, about 35 to about 50mg/kg, about 40 to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.5to about 45 mg/kg, about 0.75 to about 45 mg/kg, about 1 to about 45mg/mg, about 1.5 to about 45 mg/kb, about 2 to about 45 mg/kg, about 2.5to about 45 mg/kg, about 3 to about 45 mg/kg, about 3.5 to about 45mg/kg, about 4 to about 45 mg/kg, about 4.5 to about 45 mg/kg, about 5to about 45 mg/kg, about 7.5 to about 45 mg/kg, about 10 to about 45mg/kg, about 15 to about 45 mg/kg, about 20 to about 45 mg/kg, about 20to about 45 mg/kg, about 25 to about 45 mg/kg, about 25 to about 45mg/kg, about 30 to about 45 mg/kg, about 35 to about 45 mg/kg, about 40to about 45 mg/kg, about 0.5 to about 40 mg/kg, about 0.75 to about 40mg/kg, about 1 to about 40 mg/mg, about 1.5 to about 40 mg/kb, about 2to about 40 mg/kg, about 2.5 to about 40 mg/kg, about 3 to about 40mg/kg, about 3.5 to about 40 mg/kg, about 4 to about 40 mg/kg, about 4.5to about 40 mg/kg, about 5 to about 40 mg/kg, about 7.5 to about 40mg/kg, about 10 to about 40 mg/kg, about 15 to about 40 mg/kg, about 20to about 40 mg/kg, about 20 to about 40 mg/kg, about 25 to about 40mg/kg, about 25 to about 40 mg/kg, about 30 to about 40 mg/kg, about 35to about 40 mg/kg, about 0.5 to about 30 mg/kg, about 0.75 to about 30mg/kg, about 1 to about 30 mg/mg, about 1.5 to about 30 mg/kb, about 2to about 30 mg/kg, about 2.5 to about 30 mg/kg, about 3 to about 30mg/kg, about 3.5 to about 30 mg/kg, about 4 to about 30 mg/kg, about 4.5to about 30 mg/kg, about 5 to about 30 mg/kg, about 7.5 to about 30mg/kg, about 10 to about 30 mg/kg, about 15 to about 30 mg/kg, about 20to about 30 mg/kg, about 20 to about 30 mg/kg, about 25 to about 30mg/kg, about 0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about1 to about 20 mg/mg, about 1.5 to about 20 mg/kb, about 2 to about 20mg/kg, about 2.5 to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5to about 20 mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20mg/kg, about 5 to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10to about 20 mg/kg, or about 15 to about 20 mg/kg. In one embodiment, thedsRNA is administered at a dose of about 10 mg/kg to about 30 mg/kg.Values and ranges intermediate to the recited values are also intendedto be part of this invention.

For example, subjects can be administered a therapeutic amount of iRNA,such as about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1,3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6,4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1,6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6,7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1,9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5,13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5,20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5,27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg/kg. Valuesand ranges intermediate to the recited values are also intended to bepart of this invention.

In certain embodiments, for example, when a composition of the inventioncomprises a dsRNA as described herein and a lipid, subjects can beadministered a therapeutic amount of iRNA, such as about 0.01 mg/kg toabout 5 mg/kg, about 0.01 mg/kg to about 10 mg/kg, about 0.05 mg/kg toabout 5 mg/kg, about 0.05 mg/kg to about 10 mg/kg, about 0.1 mg/kg toabout 5 mg/kg, about 0.1 mg/kg to about 10 mg/kg, about 0.2 mg/kg toabout 5 mg/kg, about 0.2 mg/kg to about 10 mg/kg, about 0.3 mg/kg toabout 5 mg/kg, about 0.3 mg/kg to about 10 mg/kg, about 0.4 mg/kg toabout 5 mg/kg, about 0.4 mg/kg to about 10 mg/kg, about 0.5 mg/kg toabout 5 mg/kg, about 0.5 mg/kg to about 10 mg/kg, about 1 mg/kg to about5 mg/kg, about 1 mg/kg to about 10 mg/kg, about 1.5 mg/kg to about 5mg/kg, about 1.5 mg/kg to about 10 mg/kg, about 2 mg/kg to about about2.5 mg/kg, about 2 mg/kg to about 10 mg/kg, about 3 mg/kg to about 5mg/kg, about 3 mg/kg to about 10 mg/kg, about 3.5 mg/kg to about 5mg/kg, about 4 mg/kg to about 5 mg/kg, about 4.5 mg/kg to about 5 mg/kg,about 4 mg/kg to about 10 mg/kg, about 4.5 mg/kg to about 10 mg/kg,about 5 mg/kg to about 10 mg/kg, about 5.5 mg/kg to about 10 mg/kg,about 6 mg/kg to about 10 mg/kg, about 6.5 mg/kg to about 10 mg/kg,about 7 mg/kg to about 10 mg/kg, about 7.5 mg/kg to about 10 mg/kg,about 8 mg/kg to about 10 mg/kg, about 8.5 mg/kg to about 10 mg/kg,about 9 mg/kg to about 10 mg/kg, or about 9.5 mg/kg to about 10 mg/kg.Values and ranges intermediate to the recited values are also intendedto be part of this invention. For example, the dsRNA may be administeredat a dose of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1,1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6,2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6,5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1,7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6,8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about10 mg/kg. Values and ranges intermediate to the recited values are alsointended to be part of this invention.

In certain embodiments of the invention, for example, when adouble-stranded RNAi agent includes modifications (e.g., one or moremotifs of three identical modifications on three consecutivenucleotides, including one such motif at or near the cleavage site ofthe agent), six phosphorothioate linkages, and a ligand, such an agentis administered at a dose of about 0.01 to about 0.5 mg/kg, about 0.01to about 0.4 mg/kg, about 0.01 to about 0.3 mg/kg, about 0.01 to about0.2 mg/kg, about 0.01 to about 0.1 mg/kg, about 0.01 mg/kg to about 0.09mg/kg, about 0.01 mg/kg to about 0.08 mg/kg, about 0.01 mg/kg to about0.07 mg/kg, about 0.01 mg/kg to about 0.06 mg/kg, about 0.01 mg/kg toabout 0.05 mg/kg, about 0.02 to about 0.5 mg/kg, about 0.02 to about 0.4mg/kg, about 0.02 to about 0.3 mg/kg, about 0.02 to about 0.2 mg/kg,about 0.02 to about 0.1 mg/kg, about 0.02 mg/kg to about 0.09 mg/kg,about 0.02 mg/kg to about 0.08 mg/kg, about 0.02 mg/kg to about 0.07mg/kg, about 0.02 mg/kg to about 0.06 mg/kg, about 0.02 mg/kg to about0.05 mg/kg, about 0.03 to about 0.5 mg/kg, about 0.03 to about 0.4mg/kg, about 0.03 to about 0.3 mg/kg, about 0.03 to about 0.2 mg/kg,about 0.03 to about 0.1 mg/kg, about 0.03 mg/kg to about 0.09 mg/kg,about 0.03 mg/kg to about 0.08 mg/kg, about 0.03 mg/kg to about 0.07mg/kg, about 0.03 mg/kg to about 0.06 mg/kg, about 0.03 mg/kg to about0.05 mg/kg, about 0.04 to about 0.5 mg/kg, about 0.04 to about 0.4mg/kg, about 0.04 to about 0.3 mg/kg, about 0.04 to about 0.2 mg/kg,about 0.04 to about 0.1 mg/kg, about 0.04 mg/kg to about 0.09 mg/kg,about 0.04 mg/kg to about 0.08 mg/kg, about 0.04 mg/kg to about 0.07mg/kg, about 0.04 mg/kg to about 0.06 mg/kg, about 0.05 to about 0.5mg/kg, about 0.05 to about 0.4 mg/kg, about 0.05 to about 0.3 mg/kg,about 0.05 to about 0.2 mg/kg, about 0.05 to about 0.1 mg/kg, about 0.05mg/kg to about 0.09 mg/kg, about 0.05 mg/kg to about 0.08 mg/kg, orabout 0.05 mg/kg to about 0.07 mg/kg. Values and ranges intermediate tothe foregoing recited values are also intended to be part of thisinvention, e.g., the RNAi agent may be administered to the subject at adose of about 0.015 mg/kg to about 0.45 mg/mg.

For example, the RNAi agent, e.g., RNAi agent in a pharmaceuticalcomposition, may be administered at a dose of about 0.01 mg/kg, 0.0125mg/kg, 0.015 mg/kg, 0.0175 mg/kg, 0.02 mg/kg, 0.0225 mg/kg, 0.025 mg/kg,0.0275 mg/kg, 0.03 mg/kg, 0.0325 mg/kg, 0.035 mg/kg, 0.0375 mg/kg, 0.04mg/kg, 0.0425 mg/kg, 0.045 mg/kg, 0.0475 mg/kg, 0.05 mg/kg, 0.0525mg/kg, 0.055 mg/kg, 0.0575 mg/kg, 0.06 mg/kg, 0.0625 mg/kg, 0.065 mg/kg,0.0675 mg/kg, 0.07 mg/kg, 0.0725 mg/kg, 0.075 mg/kg, 0.0775 mg/kg, 0.08mg/kg, 0.0825 mg/kg, 0.085 mg/kg, 0.0875 mg/kg, 0.09 mg/kg, 0.0925mg/kg, 0.095 mg/kg, 0.0975 mg/kg, 0.1 mg/kg, 0.125 mg/kg, 0.15 mg/kg,0.175 mg/kg, 0.2 mg/kg, 0.225 mg/kg, 0.25 mg/kg, 0.275 mg/kg, 0.3 mg/kg,0.325 mg/kg, 0.35 mg/kg, 0.375 mg/kg, 0.4 mg/kg, 0.425 mg/kg, 0.45mg/kg, 0.475 mg/kg, or about 0.5 mg/kg. Values intermediate to theforegoing recited values are also intended to be part of this invention.

The pharmaceutical composition can be administered once daily, or theiRNA can be administered as two, three, or more sub-doses at appropriateintervals throughout the day or even using continuous infusion ordelivery through a controlled release formulation. In that case, theiRNA contained in each sub-dose must be correspondingly smaller in orderto achieve the total daily dosage. The dosage unit can also becompounded for delivery over several days, e.g., using a conventionalsustained release formulation which provides sustained release of theiRNA over a several day period. Sustained release formulations are wellknown in the art and are particularly useful for delivery of agents at aparticular site, such as could be used with the agents of the presentinvention. In this embodiment, the dosage unit contains a correspondingmultiple of the daily dose.

In other embodiments, a single dose of the pharmaceutical compositionscan be long lasting, such that subsequent doses are administered at notmore than 3, 4, or 5 day intervals, or at not more than 1, 2, 3, or 4week intervals. In some embodiments of the invention, a single dose ofthe pharmaceutical compositions of the invention is administered onceper week. In other embodiments of the invention, a single dose of thepharmaceutical compositions of the invention is administered bi-monthly.

The skilled artisan will appreciate that certain factors can influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual iRNAs encompassed by the inventioncan be made using conventional methodologies or on the basis of in vivotesting using an appropriate animal model, as described elsewhereherein.

Advances in mouse genetics have generated a number of mouse models forthe study of various human diseases, such as a disorder associated withiron overload that would benefit from reduction in the expression ofTMPRSS6. Such models can be used for in vivo testing of iRNA, as well asfor determining a therapeutically effective dose. Suitable mouse modelsare known in the art and include, for example, the thalassemic Th3/+mouse as a model of β-thalassemia (Douet et al., Am. J. Pathol. (2011),178(2):774-83), the HFE knockout mouse as a model of hereditaryhemochromatosis (Zhou et al. (1998) Proc. Natl. Acad. Sci USA,85:2492-2497); a Uros(mut248) mouse as a model of congenitalerythropoietic porphyria (Ged et al. (2006) Genomics, 87(1):84-92).

The pharmaceutical compositions of the present invention can beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration can be topical (e.g., by a transdermal patch), pulmonary,e.g., by inhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal, oral orparenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; subdermal, e.g., via an implanted device; or intracranial,e.g., by intraparenchymal, intrathecal or intraventricular,administration

The iRNA can be delivered in a manner to target a particular tissue,such as the liver (e.g., the hepatocytes of the liver).

Pharmaceutical compositions and formulations for topical administrationcan include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like can be necessary or desirable. Coated condoms, gloves and thelike can also be useful. Suitable topical formulations include those inwhich the iRNAs featured in the invention are in admixture with atopical delivery agent such as lipids, liposomes, fatty acids, fattyacid esters, steroids, chelating agents and surfactants. Suitable lipidsand liposomes include neutral (e.g., dioleoylphosphatidyl DOPEethanolamine, dimyristoylphosphatidyl choline DMPC,distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidylglycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAPand dioleoylphosphatidyl ethanolamine DOTMA). iRNAs featured in theinvention can be encapsulated within liposomes or can form complexesthereto, in particular to cationic liposomes. Alternatively, iRNAs canbe complexed to lipids, in particular to cationic lipids. Suitable fattyacids and esters include but are not limited to arachidonic acid, oleicacid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristicacid, palmitic acid, stearic acid, linoleic acid, linolenic acid,dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC₁₋₂₀ alkyl ester (e.g., isopropylmyristate IPM), monoglyceride,diglyceride or pharmaceutically acceptable salt thereof). Topicalformulations are described in detail in U.S. Pat. No. 6,747,014, whichis incorporated herein by reference.

A. iRNA Formulations Comprising Membranous Molecular Assemblies

An iRNA for use in the compositions and methods of the invention can beformulated for delivery in a membranous molecular assembly, e.g., aliposome or a micelle. As used herein, the term “liposome” refers to avesicle composed of amphiphilic lipids arranged in at least one bilayer,e.g., one bilayer or a plurality of bilayers. Liposomes includeunilamellar and multilamellar vesicles that have a membrane formed froma lipophilic material and an aqueous interior. The aqueous portioncontains the iRNA composition. The lipophilic material isolates theaqueous interior from an aqueous exterior, which typically does notinclude the iRNA composition, although in some examples, it may.Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomal bilayer fuses with bilayer of the cellular membranes. Asthe merging of the liposome and cell progresses, the internal aqueouscontents that include the iRNA are delivered into the cell where theiRNA can specifically bind to a target RNA and can mediate RNAi. In somecases the liposomes are also specifically targeted, e.g., to direct theiRNA to particular cell types.

A liposome containing a RNAi agent can be prepared by a variety ofmethods. In one example, the lipid component of a liposome is dissolvedin a detergent so that micelles are formed with the lipid component. Forexample, the lipid component can be an amphipathic cationic lipid orlipid conjugate. The detergent can have a high critical micelleconcentration and may be nonionic. Exemplary detergents include cholate,CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The RNAiagent preparation is then added to the micelles that include the lipidcomponent. The cationic groups on the lipid interact with the RNAi agentand condense around the RNAi agent to form a liposome. Aftercondensation, the detergent is removed, e.g., by dialysis, to yield aliposomal preparation of RNAi agent.

If necessary a carrier compound that assists in condensation can beadded during the condensation reaction, e.g., by controlled addition.For example, the carrier compound can be a polymer other than a nucleicacid (e.g., spermine or spermidine). pH can also adjusted to favorcondensation.

Methods for producing stable polynucleotide delivery vehicles, whichincorporate a polynucleotide/cationic lipid complex as structuralcomponents of the delivery vehicle, are further described in, e.g., WO96/37194, the entire contents of which are incorporated herein byreference. Liposome formation can also include one or more aspects ofexemplary methods described in Felgner, P. L. et al., Proc. Natl. Acad.Sci., USA 8:7413-7417, 1987; U.S. Pat. Nos. 4,897,355; 5,171,678;Bangham, et al. M. Mol. Biol. 23:238, 1965; Olson, et al. Biochim.Biophys. Acta 557:9, 1979; Szoka, et al. Proc. Natl. Acad. Sci. 75:4194, 1978; Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984; Kim, etal. Biochim. Biophys. Acta 728:339, 1983; and Fukunaga, et al.Endocrinol. 115:757, 1984. Commonly used techniques for preparing lipidaggregates of appropriate size for use as delivery vehicles includesonication and freeze-thaw plus extrusion (see, e.g., Mayer, et al.Biochim. Biophys. Acta 858:161, 1986). Microfluidization can be usedwhen consistently small (50 to 200 nm) and relatively uniform aggregatesare desired (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984). Thesemethods are readily adapted to packaging RNAi agent preparations intoliposomes.

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged nucleicacid molecules to form a stable complex. The positively charged nucleicacid/liposome complex binds to the negatively charged cell surface andis internalized in an endosome. Due to the acidic pH within theendosome, the liposomes are ruptured, releasing their contents into thecell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147,980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap nucleicacids rather than complex with it. Since both the nucleic acid and thelipid are similarly charged, repulsion rather than complex formationoccurs. Nevertheless, some nucleic acid is entrapped within the aqueousinterior of these liposomes. pH-sensitive liposomes have been used todeliver nucleic acids encoding the thymidine kinase gene to cellmonolayers in culture. Expression of the exogenous gene was detected inthe target cells (Zhou et al., Journal of Controlled Release, 1992, 19,269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Examples of other methods to introduce liposomes into cells in vitro andin vivo include U.S. Pat. Nos. 5,283,185; 5,171,678; WO 94/00569; WO93/24640; WO 91/16024; Felgner, J. Biol. Chem. 269:2550, 1994; Nabel,Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649,1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J. 11:417, 1992.

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporine A into different layers ofthe skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4(6) 466).

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) comprisesone or more glycolipids, such as monosialoganglioside GM1, or (B) isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., FEBS Letters,1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside GM1, galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside GM1 or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al).

In one embodiment, cationic liposomes are used. Cationic liposomespossess the advantage of being able to fuse to the cell membrane.Non-cationic liposomes, although not able to fuse as efficiently withthe plasma membrane, are taken up by macrophages in vivo and can be usedto deliver RNAi agents to macrophages.

Further advantages of liposomes include: liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated RNAi agents in their internal compartments frommetabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,”Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Importantconsiderations in the preparation of liposome formulations are the lipidsurface charge, vesicle size and the aqueous volume of the liposomes.

A positively charged synthetic cationic lipid,N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA)can be used to form small liposomes that interact spontaneously withnucleic acid to form lipid-nucleic acid complexes which are capable offusing with the negatively charged lipids of the cell membranes oftissue culture cells, resulting in delivery of RNAi agent (see, e.g.,Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 andU.S. Pat. No. 4,897,355 for a description of DOTMA and its use withDNA).

A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP)can be used in combination with a phospholipid to form DNA-complexingvesicles. Lipofectin™ Bethesda Research Laboratories, Gaithersburg, Md.)is an effective agent for the delivery of highly anionic nucleic acidsinto living tissue culture cells that comprise positively charged DOTMAliposomes which interact spontaneously with negatively chargedpolynucleotides to form complexes. When enough positively chargedliposomes are used, the net charge on the resulting complexes is alsopositive. Positively charged complexes prepared in this wayspontaneously attach to negatively charged cell surfaces, fuse with theplasma membrane, and efficiently deliver functional nucleic acids into,for example, tissue culture cells. Another commercially availablecationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane(“DOTAP”) (Boehringer Mannheim, Indianapolis, Ind.) differs from DOTMAin that the oleoyl moieties are linked by ester, rather than etherlinkages.

Other reported cationic lipid compounds include those that have beenconjugated to a variety of moieties including, for example,carboxyspermine which has been conjugated to one of two types of lipidsand includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide(“DOGS”) (Transfectam™, Promega, Madison, Wis.) anddipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”)(see, e.g., U.S. Pat. No. 5,171,678).

Another cationic lipid conjugate includes derivatization of the lipidwith cholesterol (“DC-Chol”) which has been formulated into liposomes incombination with DOPE (See, Gao, X. and Huang, L., Biochim. Biophys.Res. Commun. 179:280, 1991). Lipopolylysine, made by conjugatingpolylysine to DOPE, has been reported to be effective for transfectionin the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta1065:8, 1991). For certain cell lines, these liposomes containingconjugated cationic lipids, are said to exhibit lower toxicity andprovide more efficient transfection than the DOTMA-containingcompositions. Other commercially available cationic lipid productsinclude DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine(DOSPA) (Life Technology, Inc., Gaithersburg, Md.). Other cationiclipids suitable for the delivery of oligonucleotides are described in WO98/39359 and WO 96/37194.

Liposomal formulations are particularly suited for topicaladministration, liposomes present several advantages over otherformulations. Such advantages include reduced side effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer RNAi agent into the skin. In some implementations,liposomes are used for delivering RNAi agent to epidermal cells and alsoto enhance the penetration of RNAi agent into dermal tissues, e.g., intoskin. For example, the liposomes can be applied topically. Topicaldelivery of drugs formulated as liposomes to the skin has beendocumented (see, e.g., Weiner et al., Journal of Drug Targeting, 1992,vol. 2, 405-410 and du Plessis et al., Antiviral Research, 18, 1992,259-265; Mannino, R. J. and Fould-Fogerite, S., Biotechniques 6:682-690,1988; Itani, T. et al. Gene 56:267-276. 1987; Nicolau, C. et al. Meth.Enz. 149:157-176, 1987; Straubinger, R. M. and Papahadjopoulos, D. Meth.Enz. 101:512-527, 1983; Wang, C. Y. and Huang, L., Proc. Natl. Acad.Sci. USA 84:7851-7855, 1987).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver a drug into the dermis of mouse skin. Such formulationswith RNAi agent are useful for treating a dermatological disorder.

Liposomes that include iRNA can be made highly deformable. Suchdeformability can enable the liposomes to penetrate through pore thatare smaller than the average radius of the liposome. For example,transfersomes are a type of deformable liposomes. Transferosomes can bemade by adding surface edge activators, usually surfactants, to astandard liposomal composition. Transfersomes that include RNAi agentcan be delivered, for example, subcutaneously by infection in order todeliver RNAi agent to keratinocytes in the skin. In order to crossintact mammalian skin, lipid vesicles must pass through a series of finepores, each with a diameter less than 50 nm, under the influence of asuitable transdermal gradient. In addition, due to the lipid properties,these transferosomes can be self-optimizing (adaptive to the shape ofpores, e.g., in the skin), self-repairing, and can frequently reachtheir targets without fragmenting, and often self-loading.

Other formulations amenable to the present invention are described inU.S. provisional application Ser. No. 61/018,616, filed Jan. 2, 2008;61/018,611, filed Jan. 2, 2008; 61/039,748, filed Mar. 26, 2008;61/047,087, filed Apr. 22, 2008 and 61/051,528, filed May 8, 2008. PCTapplication no PCT/US2007/080331, filed Oct. 3, 2007 also describesformulations that are amenable to the present invention.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes can be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g., they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in Pharmaceutical Dosage Forms, MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

The iRNA for use in the methods of the invention can also be provided asmicellar formulations. “Micelles” are defined herein as a particulartype of molecular assembly in which amphipathic molecules are arrangedin a spherical structure such that all the hydrophobic portions of themolecules are directed inward, leaving the hydrophilic portions incontact with the surrounding aqueous phase. The converse arrangementexists if the environment is hydrophobic.

A mixed micellar formulation suitable for delivery through transdermalmembranes may be prepared by mixing an aqueous solution of the siRNAcomposition, an alkali metal C₈ to C₂₂ alkyl sulphate, and a micelleforming compounds. Exemplary micelle forming compounds include lecithin,hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid,glycolic acid, lactic acid, chamomile extract, cucumber extract, oleicacid, linoleic acid, linolenic acid, monoolein, monooleates,monolaurates, borage oil, evening of primrose oil, menthol, trihydroxyoxo cholanyl glycine and pharmaceutically acceptable salts thereof,glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethyleneethers and analogues thereof, polidocanol alkyl ethers and analoguesthereof, chenodeoxycholate, deoxycholate, and mixtures thereof. Themicelle forming compounds may be added at the same time or afteraddition of the alkali metal alkyl sulphate. Mixed micelles will formwith substantially any kind of mixing of the ingredients but vigorousmixing in order to provide smaller size micelles.

In one method a first micellar composition is prepared which containsthe siRNA composition and at least the alkali metal alkyl sulphate. Thefirst micellar composition is then mixed with at least three micelleforming compounds to form a mixed micellar composition. In anothermethod, the micellar composition is prepared by mixing the siRNAcomposition, the alkali metal alkyl sulphate and at least one of themicelle forming compounds, followed by addition of the remaining micelleforming compounds, with vigorous mixing.

Phenol and/or m-cresol may be added to the mixed micellar composition tostabilize the formulation and protect against bacterial growth.Alternatively, phenol and/or m-cresol may be added with the micelleforming ingredients. An isotonic agent such as glycerin may also beadded after formation of the mixed micellar composition.

For delivery of the micellar formulation as a spray, the formulation canbe put into an aerosol dispenser and the dispenser is charged with apropellant. The propellant, which is under pressure, is in liquid formin the dispenser. The ratios of the ingredients are adjusted so that theaqueous and propellant phases become one, i.e., there is one phase. Ifthere are two phases, it is necessary to shake the dispenser prior todispensing a portion of the contents, e.g., through a metered valve. Thedispensed dose of pharmaceutical agent is propelled from the meteredvalve in a fine spray.

Propellants may include hydrogen-containing chlorofluorocarbons,hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. Incertain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used.

The specific concentrations of the essential ingredients can bedetermined by relatively straightforward experimentation. For absorptionthrough the oral cavities, it is often desirable to increase, e.g., atleast double or triple, the dosage for through injection oradministration through the gastrointestinal tract.

B. Lipid Particles

iRNAs, e.g., dsRNAs of in the invention may be fully encapsulated in alipid formulation, e.g., a LNP, or other nucleic acid-lipid particle.

As used herein, the term “LNP” refers to a stable nucleic acid-lipidparticle. LNPs contain a cationic lipid, a non-cationic lipid, and alipid that prevents aggregation of the particle (e.g., a PEG-lipidconjugate). LNPs are extremely useful for systemic applications, as theyexhibit extended circulation lifetimes following intravenous (i.v.)injection and accumulate at distal sites (e.g., sites physicallyseparated from the administration site). LNPs include “pSPLP,” whichinclude an encapsulated condensing agent-nucleic acid complex as setforth in PCT Publication No. WO 00/03683. The particles of the presentinvention typically have a mean diameter of about 50 nm to about 150 nm,more typically about 60 nm to about 130 nm, more typically about 70 nmto about 110 nm, most typically about 70 nm to about 90 nm, and aresubstantially nontoxic. In addition, the nucleic acids when present inthe nucleic acid-lipid particles of the present invention are resistantin aqueous solution to degradation with a nuclease. Nucleic acid-lipidparticles and their method of preparation are disclosed in, e.g., U.S.Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; U.S.Publication No. 2010/0324120 and PCT Publication No. WO 96/40964.

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g.,lipid to dsRNA ratio) will be in the range of from about 1:1 to about50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, fromabout 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 toabout 9:1. Ranges intermediate to the above recited ranges are alsocontemplated to be part of the invention.

The cationic lipid can be, for example, N,N-dioleyl-N,N-dimethylammoniumchloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N—(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),N—(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl),1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl),1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-Dioleylamino)-1,2-propanedio (DOAP),1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA),2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) oranalogs thereof,(3aR,5s,6aS)—N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine(ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate (MC3),1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol(Tech G1), or a mixture thereof. The cationic lipid can comprise fromabout 20 mol % to about 50 mol % or about 40 mol % of the total lipidpresent in the particle.

In another embodiment, the compound2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used toprepare lipid-siRNA nanoparticles. Synthesis of2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S.provisional patent application No. 61/107,998 filed on Oct. 23, 2008,which is herein incorporated by reference. In one embodiment, thelipid-siRNA particle includes 40% 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40%Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of63.0±20 nm and a 0.027 siRNA/Lipid Ratio.

The ionizable/non-cationic lipid can be an anionic lipid or a neutrallipid including, but not limited to, distearoylphosphatidylcholine(DSPC), dioleoylphosphatidylcholine (DOPC),dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol(DOPG), dipalmitoylphosphatidylglycerol (DPPG),dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or amixture thereof. The non-cationic lipid can be from about 5 mol % toabout 90 mol %, about 10 mol %, or about 58 mol % if cholesterol isincluded, of the total lipid present in the particle.

The conjugated lipid that inhibits aggregation of particles can be, forexample, a polyethyleneglycol (PEG)-lipid including, without limitation,a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), aPEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. ThePEG-DAA conjugate can be, for example, a PEG-dilauryloxypropyl (Ci₂), aPEG-dimyristyloxypropyl (Ci₄), a PEG-dipalmityloxypropyl (Ci₆), or aPEG-distearyloxypropyl (C]₈). The conjugated lipid that preventsaggregation of particles can be from 0 mol % to about 20 mol % or about2 mol % of the total lipid present in the particle.

In some embodiments, the nucleic acid-lipid particle further includescholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol %of the total lipid present in the particle.

In one embodiment, the lipidoid ND98.4HCl (MW 1487) (see U.S. patentapplication Ser. No. 12/056,230, filed Mar. 26, 2008, which isincorporated herein by reference), Cholesterol (Sigma-Aldrich), andPEG-Ceramide C16 (Avanti Polar Lipids) can be used to preparelipid-dsRNA nanoparticles (i.e., LNP01 particles). Stock solutions ofeach in ethanol can be prepared as follows: ND98, 133 mg/ml;Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98,Cholesterol, and PEG-Ceramide C16 stock solutions can then be combinedin a, e.g., 42:48:10 molar ratio. The combined lipid solution can bemixed with aqueous dsRNA (e.g., in sodium acetate pH 5) such that thefinal ethanol concentration is about 35-45% and the final sodium acetateconcentration is about 100-300 mM. Lipid-dsRNA nanoparticles typicallyform spontaneously upon mixing. Depending on the desired particle sizedistribution, the resultant nanoparticle mixture can be extruded througha polycarbonate membrane (e.g., 100 nm cut-off) using, for example, athermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). Insome cases, the extrusion step can be omitted. Ethanol removal andsimultaneous buffer exchange can be accomplished by, for example,dialysis or tangential flow filtration. Buffer can be exchanged with,for example, phosphate buffered saline (PBS) at about pH 7, e.g., aboutpH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or aboutpH 7.4.

LNP01 formulations are described, e.g., in International ApplicationPublication No. WO 2008/042973, which is hereby incorporated byreference.

Additional exemplary lipid-dsRNA formulations are described in Table A.

TABLE A cationic lipid/non-cationic lipid/cholesterol/PEG-lipidconjugate Ionizable/Cationic Lipid Lipid:siRNA ratio LNP-11,2-Dilinolenyloxy-N,N-dimethylaminopropaneDLinDMA/DPPC/Cholesterol/PEG-cDMA (DLinDMA) (57.1/7.1/34.4/1.4)lipid:siRNA ~7:1 2-XTC 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-XTC/DPPC/Cholesterol/PEG-cDMA dioxolane (XTC) 57.1/7.1/34.4/1.4lipid:siRNA ~7:1 LNP05 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 57.5/7.5/31.5/3.5lipid:siRNA ~6:1 LNP06 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 57.5/7.5/31.5/3.5lipid:siRNA ~11:1 LNP07 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 60/7.5/31/1.5, lipid:siRNA~6:1 LNP08 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 60/7.5/31/1.5, lipid:siRNA~11:1 LNP09 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 50/10/38.5/1.5 Lipid:siRNA10:1 LNP10 (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-ALN100/DSPC/Cholesterol/PEG-DMG octadeca-9,12-dienyl)tetrahydro-3aH-50/10/38.5/1.5 cyclopenta[d][1,3]dioxol-5-amine (ALN100) Lipid:siRNA10:1 LNP11 (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-MC-3/DSPC/Cholesterol/PEG-DMG tetraen-19-yl 4-(dimethylamino)butanoate50/10/38.5/1.5 (MC3) Lipid:siRNA 10:1 LNP12 1,1′-(2-(4-(2-((2-(bis(2-Tech G1/DSPC/Cholesterol/PEG-DMG hydroxydodecyl)amino)ethyl)(2-50/10/38.5/1.5 hydroxydodecyl)amino)ethyl)piperazin-1- Lipid:siRNA 10:1yl)ethylazanediyl)didodecan-2-ol (Tech G1) LNP13 XTCXTC/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 33:1 LNP14 MC3MC3/DSPC/Chol/PEG-DMG 40/15/40/5 Lipid:siRNA: 11:1 LNP15 MC3MC3/DSPC/Chol/PEG-DSG/GalNAc-PEG-DSG 50/10/35/4.5/0.5 Lipid:siRNA: 11:1LNP16 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP17MC3 MC3/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP18 MC3MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 12:1 LNP19 MC3MC3/DSPC/Chol/PEG-DMG 50/10/35/5 Lipid:siRNA: 8:1 LNP20 MC3MC3/DSPC/Chol/PEG-DPG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP21 C12-200C12-200/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP22 XTCXTC/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1 DSPC:distearoylphosphatidylcholine DPPC: dipalmitoylphosphatidylcholinePEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avgmol wt of 2000) PEG-DSG: PEG-distyryl glycerol (C18-PEG, or PEG-C18)(PEG with avg mol wt of 2000) PEG-cDMA:PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg mol wt of 2000)

LNP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprisingformulations are described in International Publication No.WO2009/127060, filed Apr. 15, 2009, which is hereby incorporated byreference.

XTC comprising formulations are described, e.g., in U.S. ProvisionalSer. No. 61/148,366, filed Jan. 29, 2009; U.S. Provisional Ser. No.61/156,851, filed Mar. 2, 2009; U.S. Provisional Ser. No. filed Jun. 10,2009; U.S. Provisional Ser. No. 61/228,373, filed Jul. 24, 2009; U.S.Provisional Ser. No. 61/239,686, filed Sep. 3, 2009, and InternationalApplication No. PCT/US2010/022614, filed Jan. 29, 2010, which are herebyincorporated by reference.

MC3 comprising formulations are described, e.g., in U.S. Publication No.2010/0324120, filed Jun. 10, 2010, the entire contents of which arehereby incorporated by reference. ALNY-100 comprising formulations aredescribed, e.g., International patent application number PCT/US09/63933,filed on Nov. 10, 2009, which is hereby incorporated by reference.C12-200 comprising formulations are described in U.S. Provisional Ser.No. 61/175,770, filed May 5, 2009 and International Application No.PCT/US10/33777, filed May 5, 2010, which are hereby incorporated byreference.

Synthesis of Ionizable/Cationic Lipids

Any of the compounds, e.g., cationic lipids and the like, used in thenucleic acid-lipid particles of the invention can be prepared by knownorganic synthesis techniques, including the methods described in moredetail in the Examples. All substituents are as defined below unlessindicated otherwise.

“Alkyl” means a straight chain or branched, noncyclic or cyclic,saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms.Representative saturated straight chain alkyls include methyl, ethyl,n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturatedbranched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl,isopentyl, and the like. Representative saturated cyclic alkyls includecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; whileunsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, andthe like.

“Alkenyl” means an alkyl, as defined above, containing at least onedouble bond between adjacent carbon atoms. Alkenyls include both cis andtrans isomers. Representative straight chain and branched alkenylsinclude ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl,1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl,2,3-dimethyl-2-butenyl, and the like.

“Alkynyl” means any alkyl or alkenyl, as defined above, whichadditionally contains at least one triple bond between adjacent carbons.Representative straight chain and branched alkynyls include acetylenyl,propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1butynyl, and the like.

“Acyl” means any alkyl, alkenyl, or alkynyl wherein the carbon at thepoint of attachment is substituted with an oxo group, as defined below.For example, —C(═O)alkyl, —C(═O)alkenyl, and —C(═O)alkynyl are acylgroups.

“Heterocycle” means a 5- to 7-membered monocyclic, or 7- to 10-memberedbicyclic, heterocyclic ring which is either saturated, unsaturated, oraromatic, and which contains from 1 or 2 heteroatoms independentlyselected from nitrogen, oxygen and sulfur, and wherein the nitrogen andsulfur heteroatoms can be optionally oxidized, and the nitrogenheteroatom can be optionally quaternized, including bicyclic rings inwhich any of the above heterocycles are fused to a benzene ring. Theheterocycle can be attached via any heteroatom or carbon atom.Heterocycles include heteroaryls as defined below. Heterocycles includemorpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl,hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl,tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

The terms “optionally substituted alkyl”, “optionally substitutedalkenyl”, “optionally substituted alkynyl”, “optionally substitutedacyl”, and “optionally substituted heterocycle” means that, whensubstituted, at least one hydrogen atom is replaced with a substituent.In the case of an oxo substituent (═O) two hydrogen atoms are replaced.In this regard, substituents include oxo, halogen, heterocycle, —CN,—ORx, —NRxRy, —NRxC(═O)Ry, —NRxSO2Ry, —C(═O)Rx, —C(═O)ORx, —C(═O)NRxRy,—SOnRx and —SOnNRxRy, wherein n is 0, 1 or 2, Rx and Ry are the same ordifferent and independently hydrogen, alkyl or heterocycle, and each ofsaid alkyl and heterocycle substituents can be further substituted withone or more of oxo, halogen, —OH, —CN, alkyl, —ORx, heterocycle, —NRxRy,—NRxC(═O)Ry, —NRxSO2Ry, —C(═O)Rx, —C(═O)ORx, —C(═O)NRxRy, —SOnRx and—SOnNRxRy.

“Halogen” means fluoro, chloro, bromo and iodo.

In some embodiments, the methods of the invention can require the use ofprotecting groups. Protecting group methodology is well known to thoseskilled in the art (see, for example, Protective Groups in OrganicSynthesis, Green, T. W. et al., Wiley-Interscience, New York City,1999). Briefly, protecting groups within the context of this inventionare any group that reduces or eliminates unwanted reactivity of afunctional group. A protecting group can be added to a functional groupto mask its reactivity during certain reactions and then removed toreveal the original functional group. In some embodiments an “alcoholprotecting group” is used. An “alcohol protecting group” is any groupwhich decreases or eliminates unwanted reactivity of an alcoholfunctional group. Protecting groups can be added and removed usingtechniques well known in the art.

Synthesis of Formula A

In some embodiments, nucleic acid-lipid particles of the invention areformulated using a cationic lipid of formula A:

where R1 and R2 are independently alkyl, alkenyl or alkynyl, each can beoptionally substituted, and R3 and R4 are independently lower alkyl orR3 and R4 can be taken together to form an optionally substitutedheterocyclic ring. In some embodiments, the cationic lipid is XTC(2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane). In general, thelipid of formula A above can be made by the following Reaction Schemes 1or 2, wherein all substituents are as defined above unless indicatedotherwise.

Lipid A, where R1 and R2 are independently alkyl, alkenyl or alkynyl,each can be optionally substituted, and R3 and R4 are independentlylower alkyl or R3 and R4 can be taken together to form an optionallysubstituted heterocyclic ring, can be prepared according to Scheme 1.Ketone 1 and bromide 2 can be purchased or prepared according to methodsknown to those of ordinary skill in the art. Reaction of 1 and 2 yieldsketal 3. Treatment of ketal 3 with amine 4 yields lipids of formula A.The lipids of formula A can be converted to the corresponding ammoniumsalt with an organic salt of formula 5, where X is anion counter ionselected from halogen, hydroxide, phosphate, sulfate, or the like.

Alternatively, the ketone 1 starting material can be prepared accordingto Scheme 2. Grignard reagent 6 and cyanide 7 can be purchased orprepared according to methods known to those of ordinary skill in theart. Reaction of 6 and 7 yields ketone 1. Conversion of ketone 1 to thecorresponding lipids of formula A is as described in Scheme 1.

Synthesis of MC3

Preparation of DLin-M-C3-DMA (i.e.,(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate) was as follows. A solution of(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (0.53 g),4-N,N-dimethylaminobutyric acid hydrochloride (0.51 g),4-N,N-dimethylaminopyridine (0.61 g) and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.53 g) indichloromethane (5 mL) was stirred at room temperature overnight. Thesolution was washed with dilute hydrochloric acid followed by diluteaqueous sodium bicarbonate. The organic fractions were dried overanhydrous magnesium sulphate, filtered and the solvent removed on arotovap. The residue was passed down a silica gel column (20 g) using a1-5% methanol/dichloromethane elution gradient. Fractions containing thepurified product were combined and the solvent removed, yielding acolorless oil (0.54 g).

Synthesis of ALNY-100

Synthesis of ketal 519 [ALNY-100] was performed using the followingscheme 3:

Synthesis of 515

To a stirred suspension of LiAlH4 (3.74 g, 0.09852 mol) in 200 mlanhydrous THF in a two neck RBF (1 L), was added a solution of 514 (10g, 0.04926 mol) in 70 mL of THF slowly at 0° C. under nitrogenatmosphere. After complete addition, reaction mixture was warmed to roomtemperature and then heated to reflux for 4 h. Progress of the reactionwas monitored by TLC. After completion of reaction (by TLC) the mixturewas cooled to 0° C. and quenched with careful addition of saturatedNa2SO4 solution. Reaction mixture was stirred for 4 h at roomtemperature and filtered off. Residue was washed well with THF. Thefiltrate and washings were mixed and diluted with 400 mL dioxane and 26mL conc. HCl and stirred for 20 minutes at room temperature. Thevolatilities were stripped off under vacuum to furnish the hydrochloridesalt of 515 as a white solid. Yield: 7.12 g 1H-NMR (DMSO, 400 MHz):δ=9.34 (broad, 2H), 5.68 (s, 2H), 3.74 (m, 1H), 2.66-2.60 (m, 2H),2.50-2.45 (m, 5H).

Synthesis of 516

To a stirred solution of compound 515 in 100 mL dry DCM in a 250 mL twoneck RBF, was added NEt3 (37.2 mL, 0.2669 mol) and cooled to 0° C. undernitrogen atmosphere. After a slow addition ofN-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL dryDCM, reaction mixture was allowed to warm to room temperature. Aftercompletion of the reaction (2-3 h by TLC) mixture was washedsuccessively with 1N HCl solution (1×100 mL) and saturated NaHCO3solution (1×50 mL). The organic layer was then dried over anhyd. Na2SO4and the solvent was evaporated to give crude material which was purifiedby silica gel column chromatography to get 516 as sticky mass. Yield: 11g (89%). 1H-NMR (CDCl3, 400 MHz): δ=7.36-7.27 (m, 5H), 5.69 (s, 2H),5.12 (s, 2H), 4.96 (br., 1H) 2.74 (s, 3H), 2.60 (m, 2H), 2.30-2.25 (m,2H). LC-MS [M+H] −232.3 (96.94%).

Synthesis of 517A and 517B

The cyclopentene 516 (5 g, 0.02164 mol) was dissolved in a solution of220 mL acetone and water (10:1) in a single neck 500 mL RBF and to itwas added N-methyl morpholine-N-oxide (7.6 g, 0.06492 mol) followed by4.2 mL of 7.6% solution of OsO4 (0.275 g, 0.00108 mol) in tert-butanolat room temperature. After completion of the reaction (˜3 h), themixture was quenched with addition of solid Na2SO3 and resulting mixturewas stirred for 1.5 h at room temperature. Reaction mixture was dilutedwith DCM (300 mL) and washed with water (2×100 mL) followed by saturatedNaHCO₃ (1×50 mL) solution, water (1×30 mL) and finally with brine (lx 50mL). Organic phase was dried over an.Na2SO4 and solvent was removed invacuum. Silica gel column chromatographic purification of the crudematerial was afforded a mixture of diastereomers, which were separatedby prep HPLC. Yield: −6 g crude 517A—Peak-1 (white solid), 5.13 g (96%).1H-NMR (DMSO, 400 MHz): δ=7.39-7.31 (m, 5H), 5.04 (s, 2H), 4.78-4.73 (m,1H), 4.48-4.47 (d, 2H), 3.94-3.93 (m, 2H), 2.71 (s, 3H), 1.72-1.67 (m,4H). LC-MS—[M+H] −266.3, [M+NH4+]− 283.5 present, HPLC-97.86%.Stereochemistry confirmed by X-ray.

Synthesis of 518

Using a procedure analogous to that described for the synthesis ofcompound 505, compound 518 (1.2 g, 41%) was obtained as a colorless oil.1H-NMR (CDCl3, 400 MHz): δ=7.35-7.33 (m, 4H), 7.30-7.27 (m, 1H),5.37-5.27 (m, 8H), 5.12 (s, 2H), 4.75 (m, 1H), 4.58-4.57 (m, 2H),2.78-2.74 (m, 7H), 2.06-2.00 (m, 8H), 1.96-1.91 (m, 2H), 1.62 (m, 4H),1.48 (m, 2H), 1.37-1.25 (br m, 36H), 0.87 (m, 6H). HPLC-98.65%.

General Procedure for the Synthesis of Compound 519

A solution of compound 518 (1 eq) in hexane (15 mL) was added in adrop-wise fashion to an ice-cold solution of LAH in THF (1 M, 2 eq).After complete addition, the mixture was heated at 400° C. over 0.5 hthen cooled again on an ice bath. The mixture was carefully hydrolyzedwith saturated aqueous Na2SO4 then filtered through celite and reducedto an oil. Column chromatography provided the pure 519 (1.3 g, 68%)which was obtained as a colorless oil. 13C NMR δ=130.2, 130.1 (×2),127.9 (×3), 112.3, 79.3, 64.4, 44.7, 38.3, 35.4, 31.5, 29.9 (×2), 29.7,29.6 (×2), 29.5 (×3), 29.3 (×2), 27.2 (×3), 25.6, 24.5, 23.3, 226, 14.1;Electrospray MS (+ve): Molecular weight for C44H80NO2 (M+H)+ Calc.654.6, Found 654.6.

Formulations prepared by either the standard or extrusion-free methodcan be characterized in similar manners. For example, formulations aretypically characterized by visual inspection. They should be whitishtranslucent solutions free from aggregates or sediment. Particle sizeand particle size distribution of lipid-nanoparticles can be measured bylight scattering using, for example, a Malvern Zetasizer Nano ZS(Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nmin size. The particle size distribution should be unimodal. The totaldsRNA concentration in the formulation, as well as the entrappedfraction, is estimated using a dye exclusion assay. A sample of theformulated dsRNA can be incubated with an RNA-binding dye, such asRibogreen (Molecular Probes) in the presence or absence of a formulationdisrupting surfactant, e.g., 0.5% Triton-X100. The total dsRNA in theformulation can be determined by the signal from the sample containingthe surfactant, relative to a standard curve. The entrapped fraction isdetermined by subtracting the “free” dsRNA content (as measured by thesignal in the absence of surfactant) from the total dsRNA content.Percent entrapped dsRNA is typically >85%. For LNP formulation, theparticle size is at least 30 nm, at least 40 nm, at least 50 nm, atleast 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least100 nm, at least 110 nm, and at least 120 nm. The suitable range istypically about at least 50 nm to about at least 110 nm, about at least60 nm to about at least 100 nm, or about at least 80 nm to about atleast 90 nm.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders can be desirable. In some embodiments, oralformulations are those in which dsRNAs featured in the invention areadministered in conjunction with one or more penetration enhancersurfactants and chelators. Suitable surfactants include fatty acidsand/or esters or salts thereof, bile acids and/or salts thereof.Suitable bile acids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitablefatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g., sodium). In some embodiments, combinations of penetrationenhancers are used, for example, fatty acids/salts in combination withbile acids/salts. One exemplary combination is the sodium salt of lauricacid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAsfeatured in the invention can be delivered orally, in granular formincluding sprayed dried particles, or complexed to form micro ornanoparticles. DsRNA complexing agents include poly-amino acids;polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Suitable complexing agents include chitosan,N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine,polyspermines, protamine, polyvinylpyridine,polythiodiethylaminomethylethylene P (TDAE), polyaminostyrene (e.g.,p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor dsRNAs and their preparation are described in detail in U.S. Pat.No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No. 6,747,014,each of which is incorporated herein by reference.

Compositions and formulations for parenteral, intraparenchymal (into thebrain), intrathecal, intraventricular or intrahepatic administration caninclude sterile aqueous solutions which can also contain buffers,diluents and other suitable additives such as, but not limited to,penetration enhancers, carrier compounds and other pharmaceuticallyacceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions can be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids. Particularlypreferred are formulations that target the liver when treating hepaticdisorders such as hepatic carcinoma.

The pharmaceutical formulations of the present invention, which canconveniently be presented in unit dosage form, can be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention can be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention can also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions can further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension can also contain stabilizers.

C. Additional Formulations

Emulsions

The compositions of the present invention can be prepared and formulatedas emulsions. Emulsions are typically heterogeneous systems of oneliquid dispersed in another in the form of droplets usually exceeding0.1 μm in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms andDrug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al.,in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,Pa., 1985, p. 301). Emulsions are often biphasic systems comprising twoimmiscible liquid phases intimately mixed and dispersed with each other.In general, emulsions can be of either the water-in-oil (w/o) or theoil-in-water (o/w) variety. When an aqueous phase is finely divided intoand dispersed as minute droplets into a bulk oily phase, the resultingcomposition is called a water-in-oil (w/o) emulsion. Alternatively, whenan oily phase is finely divided into and dispersed as minute dropletsinto a bulk aqueous phase, the resulting composition is called anoil-in-water (o/w) emulsion. Emulsions can contain additional componentsin addition to the dispersed phases, and the active drug which can bepresent as a solution in either the aqueous phase, oily phase or itselfas a separate phase. Pharmaceutical excipients such as emulsifiers,stabilizers, dyes, and anti-oxidants can also be present in emulsions asneeded. Pharmaceutical emulsions can also be multiple emulsions that arecomprised of more than two phases such as, for example, in the case ofoil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.Such complex formulations often provide certain advantages that simplebinary emulsions do not. Multiple emulsions in which individual oildroplets of an o/w emulsion enclose small water droplets constitute aw/o/w emulsion. Likewise a system of oil droplets enclosed in globulesof water stabilized in an oily continuous phase provides an o/w/oemulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion can be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatcan be incorporated into either phase of the emulsion. Emulsifiers canbroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug DeliverySystems, Allen, L V., Popovich N G., and Ansel H C., 2004, LippincottWilliams & Wilkins (8th ed.), New York, N.Y.; Idson, in PharmaceuticalDosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199).Surfactants are typically amphiphilic and comprise a hydrophilic and ahydrophobic portion. The ratio of the hydrophilic to the hydrophobicnature of the surfactant has been termed the hydrophile/lipophilebalance (HLB) and is a valuable tool in categorizing and selectingsurfactants in the preparation of formulations. Surfactants can beclassified into different classes based on the nature of the hydrophilicgroup: nonionic, anionic, cationic and amphoteric (see e.g., Ansel'sPharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V.,Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8thed.), New York, N.Y. Rieger, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that can readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used can be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature (see e.g., Ansel's Pharmaceutical Dosage Forms andDrug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsionformulations for oral delivery have been very widely used because ofease of formulation, as well as efficacy from an absorption andbioavailability standpoint (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritivepreparations are among the materials that have commonly beenadministered orally as o/w emulsions.

ii. Microemulsions

In one embodiment of the present invention, the compositions of iRNAsand nucleic acids are formulated as microemulsions. A microemulsion canbe defined as a system of water, oil and amphiphile which is a singleoptically isotropic and thermodynamically stable liquid solution (seee.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems,Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams &Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical DosageForms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc.,New York, N.Y., volume 1, p. 245). Typically microemulsions are systemsthat are prepared by first dispersing an oil in an aqueous surfactantsolution and then adding a sufficient amount of a fourth component,generally an intermediate chain-length alcohol to form a transparentsystem. Therefore, microemulsions have also been described asthermodynamically stable, isotropically clear dispersions of twoimmiscible liquids that are stabilized by interfacial films ofsurface-active molecules (Leung and Shah, in: Controlled Release ofDrugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCHPublishers, New York, pages 185-215). Microemulsions commonly areprepared via a combination of three to five components that include oil,water, surfactant, cosurfactant and electrolyte. Whether themicroemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) typeis dependent on the properties of the oil and surfactant used and on thestructure and geometric packing of the polar heads and hydrocarbon tailsof the surfactant molecules (Schott, in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (see e.g.,Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins(8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 335). Compared to conventional emulsions,microemulsions offer the advantage of solubilizing water-insoluble drugsin a formulation of thermodynamically stable droplets that are formedspontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions can, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase can typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase can include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos.6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (see e.g., U.S.Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides etal., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci.,1996, 85, 138-143). Often microemulsions can form spontaneously whentheir components are brought together at ambient temperature. This canbe particularly advantageous when formulating thermolabile drugs,peptides or iRNAs. Microemulsions have also been effective in thetransdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of iRNAs and nucleic acids from thegastrointestinal tract, as well as improve the local cellular uptake ofiRNAs and nucleic acids.

Microemulsions of the present invention can also contain additionalcomponents and additives such as sorbitan monostearate (Grill 3),Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the iRNAs and nucleic acidsof the present invention. Penetration enhancers used in themicroemulsions of the present invention can be classified as belongingto one of five broad categories—surfactants, fatty acids, bile salts,chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

iii. Microparticles

An RNAi agent of the invention may be incorporated into a particle,e.g., a microparticle. Microparticles can be produced by spray-drying,but may also be produced by other methods including lyophilization,evaporation, fluid bed drying, vacuum drying, or a combination of thesetechniques.

iv. Penetration Enhancers

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly iRNAs, to the skin of animals. Most drugs are present insolution in both ionized and nonionized forms. However, usually onlylipid soluble or lipophilic drugs readily cross cell membranes. It hasbeen discovered that even non-lipophilic drugs can cross cell membranesif the membrane to be crossed is treated with a penetration enhancer. Inaddition to aiding the diffusion of non-lipophilic drugs across cellmembranes, penetration enhancers also enhance the permeability oflipophilic drugs.

Penetration enhancers can be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (see e.g., Malmsten, M.Surfactants and polymers in drug delivery, Informa Health Care, NewYork, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic DrugCarrier Systems, 1991, p. 92). Each of the above mentioned classes ofpenetration enhancers are described below in greater detail.

Surfactants (or “surface-active agents”) are chemical entities which,when dissolved in an aqueous solution, reduce the surface tension of thesolution or the interfacial tension between the aqueous solution andanother liquid, with the result that absorption of iRNAs through themucosa is enhanced. In addition to bile salts and fatty acids, thesepenetration enhancers include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (seee.g., Malmsten, M. Surfactants and polymers in drug delivery, InformaHealth Care, New York, N.Y., 2002; Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemicalemulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988,40, 252).

Various fatty acids and their derivatives which act as penetrationenhancers include, for example, oleic acid, lauric acid, capric acid(n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleicacid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C₁₋₂₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (see e.g.,Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers,Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic DrugCarrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol.,1992, 44, 651-654).

The physiological role of bile includes the facilitation of dispersionand absorption of lipids and fat-soluble vitamins (see e.g., Malmsten,M. Surfactants and polymers in drug delivery, Informa Health Care, NewYork, N.Y., 2002; Brunton, Chapter 38 in: Goodman & Gilman's ThePharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds.,McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts,and their synthetic derivatives, act as penetration enhancers. Thus theterm “bile salts” includes any of the naturally occurring components ofbile as well as any of their synthetic derivatives. Suitable bile saltsinclude, for example, cholic acid (or its pharmaceutically acceptablesodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (see e.g.,Malmsten, M. Surfactants and polymers in drug delivery, Informa HealthCare, New York, N.Y., 2002; Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In:Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto etal., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm.Sci., 1990, 79, 579-583).

Chelating agents, as used in connection with the present invention, canbe defined as compounds that remove metallic ions from solution byforming complexes therewith, with the result that absorption of iRNAsthrough the mucosa is enhanced. With regards to their use as penetrationenhancers in the present invention, chelating agents have the addedadvantage of also serving as DNase inhibitors, as most characterized DNAnucleases require a divalent metal ion for catalysis and are thusinhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618,315-339). Suitable chelating agents include but are not limited todisodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates(e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acylderivatives of collagen, laureth-9 and N-amino acyl derivatives ofbeta-diketones (enamines)(see e.g., Katdare, A. et al., Excipientdevelopment for pharmaceutical, biotechnology, and drug delivery, CRCPress, Danvers, Mass., 2006; Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. ControlRel., 1990, 14, 43-51).

As used herein, non-chelating non-surfactant penetration enhancingcompounds can be defined as compounds that demonstrate insignificantactivity as chelating agents or as surfactants but that nonethelessenhance absorption of iRNAs through the alimentary mucosa (see e.g.,Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33). This class of penetration enhancers includes, for example,unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanonederivatives (Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, page 92); and non-steroidal anti-inflammatory agents suchas diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al.,J. Pharm. Pharmacol., 1987, 39, 621-626).

Agents that enhance uptake of iRNAs at the cellular level can also beadded to the pharmaceutical and other compositions of the presentinvention. For example, cationic lipids, such as lipofectin (Junichi etal, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, andpolycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof dsRNAs. Examples of commercially available transfection reagentsinclude, for example Lipofectamine™ (Invitrogen; Carlsbad, Calif.),Lipofectamine 2000™ (Invitrogen; Carlsbad, Calif.), 293Fectin™(Invitrogen; Carlsbad, Calif.), Cellfectin™ (Invitrogen; Carlsbad,Calif.), DMRIE-C™ (Invitrogen; Carlsbad, Calif.), FreeStyle™ MAX(Invitrogen; Carlsbad, Calif.), Lipofectamine™ 2000 CD (Invitrogen;Carlsbad, Calif.), Lipofectamine™ (Invitrogen; Carlsbad, Calif.),RNAiMAX (Invitrogen; Carlsbad, Calif.), Oligofectamine™ (Invitrogen;Carlsbad, Calif.), Optifect™ (Invitrogen; Carlsbad, Calif.), X-tremeGENEQ2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAPLiposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPERLiposomal Transfection Reagent (Grenzacherstrasse, Switzerland), orFugene (Grenzacherstrasse, Switzerland), Transfectam® Reagent (Promega;Madison, Wis.), TransFast™ Transfection Reagent (Promega; Madison,Wis.), Tfx™-20 Reagent (Promega; Madison, Wis.), Tfx™-50 Reagent(Promega; Madison, Wis.), DreamFect™ (OZ Biosciences; Marseille,France), EcoTransfect (OZ Biosciences; Marseille, France), TransPass^(a)D1 Transfection Reagent (New England Biolabs; Ipswich, Mass., USA),LyoVec™/LipoGen™ (Invitrogen; San Diego, Calif., USA), PerFectinTransfection Reagent (Genlantis; San Diego, Calif., USA), NeuroPORTERTransfection Reagent (Genlantis; San Diego, Calif., USA), GenePORTERTransfection reagent (Genlantis; San Diego, Calif., USA), GenePORTER 2Transfection reagent (Genlantis; San Diego, Calif., USA), CytofectinTransfection Reagent (Genlantis; San Diego, Calif., USA), BaculoPORTERTransfection Reagent (Genlantis; San Diego, Calif., USA), TroganPORTER™transfection Reagent (Genlantis; San Diego, Calif., USA), RiboFect(Bioline; Taunton, Mass., USA), PlasFect (Bioline; Taunton, Mass., USA),UniFECTOR (B-Bridge International; Mountain View, Calif., USA),SureFECTOR (B-Bridge International; Mountain View, Calif., USA), orHiFect™ (B-Bridge International, Mountain View, Calif., USA), amongothers.

Other agents can be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

v. Carriers

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The coadministration of a nucleic acid and a carriercompound, typically with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extracirculatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioate dsRNA in hepatic tissue can be reduced when it iscoadministered with polyinosinic acid, dextran sulfate, polycytidic acidor 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao etal., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl.Acid Drug Dev., 1996, 6, 177-183.

vi. Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient can be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc).

Pharmaceutically acceptable organic or inorganic excipients suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can also be used to formulate the compositions of thepresent invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids can includesterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions of the nucleic acids inliquid or solid oil bases. The solutions can also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

vii. Other Components

The compositions of the present invention can additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions can contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or can contain additionalmaterials useful in physically formulating various dosage forms of thecompositions of the present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Aqueous suspensions can contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension can also contain stabilizers.

In some embodiments, pharmaceutical compositions featured in theinvention include (a) one or more iRNA compounds and (b) one or moreagents which function by a non-RNAi mechanism and which are useful intreating a bleeding disorder. Examples of such agents include, but arenot limited to an anti-inflammatory agent, anti-steatosis agent,anti-viral, and/or anti-fibrosis agent. In addition, other substancescommonly used to protect the liver, such as silymarin, can also be usedin conjunction with the iRNAs described herein. Other agents useful fortreating liver diseases include telbivudine, entecavir, and proteaseinhibitors such as telaprevir and other disclosed, for example, in Tunget al., U.S. Application Publication Nos. 2005/0148548, 2004/0167116,and 2003/0144217; and in Hale et al., U.S. Application Publication No.2004/0127488.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofcompositions featured herein in the invention lies generally within arange of circulating concentrations that include the ED50 with little orno toxicity. The dosage can vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the methods featured in the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose can be formulated in animal models to achieve acirculating plasma concentration range of the compound or, whenappropriate, of the polypeptide product of a target sequence (e.g.,achieving a decreased concentration of the polypeptide) that includesthe IC₅₀ (i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma can be measured, for example, by highperformance liquid chromatography.

In addition to their administration, as discussed above, the iRNAsfeatured in the invention can be administered in combination with otherknown agents effective in treatment of pathological processes that aremediated by iron overload and that can be treated by inhibiting TMPRSS6expression. In any event, the administering physician can adjust theamount and timing of iRNA administration on the basis of resultsobserved using standard measures of efficacy known in the art ordescribed herein.

V. Methods for Inhibiting TMPRSS6 Expression

The present invention provides methods of inhibiting expression ofTMPRSS6 (matriptase-2) in a cell. The methods include contacting a cellwith an RNAi agent, e.g., a double stranded RNAi agent, in an amounteffective to inhibit expression of the TMPRSS6 in the cell, therebyinhibiting expression of the TMPRSS6 in the cell.

Contacting of a cell with a double stranded RNAi agent may be done invitro or in vivo. Contacting a cell in vivo with the RNAi agent includescontacting a cell or group of cells within a subject, e.g., a humansubject, with the RNAi agent. Combinations of in vitro and in vivomethods of contacting are also possible. Contacting may be direct orindirect, as discussed above. Furthermore, contacting a cell may beaccomplished via a targeting ligand, including any ligand describedherein or known in the art. In preferred embodiments, the targetingligand is a carbohydrate moiety, e.g., a GalNAc₃ ligand, or any otherligand that directs the RNAi agent to a site of interest, e.g., theliver of a subject.

The term “inhibiting,” as used herein, is used interchangeably with“reducing,” “silencing,” “downregulating” and other similar terms, andincludes any level of inhibition.

The phrase “inhibiting expression of a TMPRSS6” is intended to refer toinhibition of expression of any TMPRSS6 gene (such as, e.g., a mouseTMPRSS6 gene, a rat TMPRSS6 gene, a monkey TMPRSS6 gene, or a humanTMPRSS6 gene) as well as variants or mutants of a TMPRSS6 gene. Thus,the TMPRSS6 gene may be a wild-type TMPRSS6 gene, a mutant TMPRSS6 gene,or a transgenic TMPRSS6 gene in the context of a genetically manipulatedcell, group of cells, or organism.

“Inhibiting expression of a TMPRSS6 gene” includes any level ofinhibition of a TMPRSS6 gene, e.g., at least partial suppression of theexpression of a TMPRSS6 gene. The expression of the TMPRSS6 gene may beassessed based on the level, or the change in the level, of any variableassociated with TMPRSS6 gene expression, e.g., TMPRSS6 mRNA level,TMPRSS6 protein level, or lipid levels. This level may be assessed in anindividual cell or in a group of cells, including, for example, a samplederived from a subject.

Inhibition may be assessed by a decrease in an absolute or relativelevel of one or more variables that are associated with TMPRSS6expression compared with a control level. The control level may be anytype of control level that is utilized in the art, e.g., a pre-dosebaseline level, or a level determined from a similar subject, cell, orsample that is untreated or treated with a control (such as, e.g.,buffer only control or inactive agent control).

In some embodiments of the methods of the invention, expression of aTMPRSS6 gene is inhibited by at least about 5%, at least about 10%, atleast about 15%, at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 55%, at least about 60%, at least about65%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%. at least about 95%, atleast about 96%, at least about 97%, at least about 98%, or at leastabout 99%.

Inhibition of the expression of a TMPRSS6 gene may be manifested by areduction of the amount of mRNA expressed by a first cell or group ofcells (such cells may be present, for example, in a sample derived froma subject) in which a TMPRSS6 gene is transcribed and which has or havebeen treated (e.g., by contacting the cell or cells with an RNAi agentof the invention, or by administering an RNAi agent of the invention toa subject in which the cells are or were present) such that theexpression of a TMPRSS6 gene is inhibited, as compared to a second cellor group of cells substantially identical to the first cell or group ofcells but which has not or have not been so treated (control cell(s)).In preferred embodiments, the inhibition is assessed by expressing thelevel of mRNA in treated cells as a percentage of the level of mRNA incontrol cells, using the following formula:

${\frac{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right) - \left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {treated}\mspace{14mu} {cells}} \right)}{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right)} \cdot 100}\%$

Alternatively, inhibition of the expression of a TMPRSS6 gene may beassessed in terms of a reduction of a parameter that is functionallylinked to TMPRSS6 gene expression, e.g., TMPRSS6 protein expression,hepcidin gene or protein expression, or iron levels in tissues or serum.TMPRSS6 gene silencing may be determined in any cell expressing TMPRSS6,either constitutively or by genomic engineering, and by any assay knownin the art. The liver is the major site of TMPRSS6 expression. Othersignificant sites of expression include the kidneys and the uterus.

Inhibition of the expression of a TMPRSS6 protein may be manifested by areduction in the level of the TMPRSS6 protein that is expressed by acell or group of cells (e.g., the level of protein expressed in a samplederived from a subject). As explained above for the assessment of mRNAsuppression, the inhibition of protein expression levels in a treatedcell or group of cells may similarly be expressed as a percentage of thelevel of protein in a control cell or group of cells.

A control cell or group of cells that may be used to assess theinhibition of the expression of a TMPRSS6 gene includes a cell or groupof cells that has not yet been contacted with an RNAi agent of theinvention. For example, the control cell or group of cells may bederived from an individual subject (e.g., a human or animal subject)prior to treatment of the subject with an RNAi agent.

The level of TMPRSS6 mRNA that is expressed by a cell or group of cellsmay be determined using any method known in the art for assessing mRNAexpression. In one embodiment, the level of expression of TMPRSS6 in asample is determined by detecting a transcribed polynucleotide, orportion thereof, e.g., mRNA of the TMPRSS6 gene. RNA may be extractedfrom cells using RNA extraction techniques including, for example, usingacid phenol/guanidine isothiocyanate extraction (RNAzol B; Biogenesis),RNeasy RNA preparation kits (Qiagen) or PAXgene (PreAnalytix,Switzerland). Typical assay formats utilizing ribonucleic acidhybridization include nuclear run-on assays, RT-PCR, RNase protectionassays (Melton et al., Nuc. Acids Res. 12:7035), Northern blotting, insitu hybridization, and microarray analysis.

In one embodiment, the level of expression of TMPRSS6 is determinedusing a nucleic acid probe. The term “probe”, as used herein, refers toany molecule that is capable of selectively binding to a specificTMPRSS6. Probes can be synthesized by one of skill in the art, orderived from appropriate biological preparations. Probes may bespecifically designed to be labeled. Examples of molecules that can beutilized as probes include, but are not limited to, RNA, DNA, proteins,antibodies, and organic molecules.

Isolated mRNA can be used in hybridization or amplification assays thatinclude, but are not limited to, Southern or Northern analyses,polymerase chain reaction (PCR) analyses and probe arrays. One methodfor the determination of mRNA levels involves contacting the isolatedmRNA with a nucleic acid molecule (probe) that can hybridize to TMPRSS6mRNA. In one embodiment, the mRNA is immobilized on a solid surface andcontacted with a probe, for example by running the isolated mRNA on anagarose gel and transferring the mRNA from the gel to a membrane, suchas nitrocellulose. In an alternative embodiment, the probe(s) areimmobilized on a solid surface and the mRNA is contacted with theprobe(s), for example, in an Affymetrix gene chip array. A skilledartisan can readily adapt known mRNA detection methods for use indetermining the level of TMPRSS6 mRNA.

An alternative method for determining the level of expression of TMPRSS6in a sample involves the process of nucleic acid amplification and/orreverse transcriptase (to prepare cDNA) of for example mRNA in thesample, e.g., by RT-PCR (the experimental embodiment set forth inMullis, 1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany(1991) Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequencereplication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh et al. (1989)Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi etal. (1988) Bio/Technology 6:1197), rolling circle replication (Lizardiet al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplificationmethod, followed by the detection of the amplified molecules usingtechniques well known to those of skill in the art. These detectionschemes are especially useful for the detection of nucleic acidmolecules if such molecules are present in very low numbers. Inparticular aspects of the invention, the level of expression of TMPRSS6is determined by quantitative fluorogenic RT-PCR (i.e., the TaqMan™System).

The expression levels of TMPRSS6 mRNA may be monitored using a membraneblot (such as used in hybridization analysis such as Northern, Southern,dot, and the like), or microwells, sample tubes, gels, beads or fibers(or any solid support comprising bound nucleic acids). See U.S. Pat.Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934, which areincorporated herein by reference. The determination of TMPRSS6expression level may also comprise using nucleic acid probes insolution.

In preferred embodiments, the level of mRNA expression is assessed usingbranched DNA (bDNA) assays or real time PCR (qPCR). The use of thesemethods is described and exemplified in the Examples presented herein.

The level of TMPRSS6 protein expression may be determined using anymethod known in the art for the measurement of protein levels. Suchmethods include, for example, electrophoresis, capillaryelectrophoresis, high performance liquid chromatography (HPLC), thinlayer chromatography (TLC), hyperdiffusion chromatography, fluid or gelprecipitin reactions, absorption spectroscopy, a colorimetric assays,spectrophotometric assays, flow cytometry, immunodiffusion (single ordouble), immunoelectrophoresis, Western blotting, radioimmunoassay(RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescentassays, electrochemiluminescence assays, and the like.

The term “sample” as used herein refers to a collection of similarfluids, cells, or tissues isolated from a subject, as well as fluids,cells, or tissues present within a subject. Examples of biologicalfluids include blood, serum and serosal fluids, plasma, lymph, urine,cerebrospinal fluid, saliva, ocular fluids, and the like. Tissue samplesmay include samples from tissues, organs or localized regions. Forexample, samples may be derived from particular organs, parts of organs,or fluids or cells within those organs. In certain embodiments, samplesmay be derived from the liver (e.g., whole liver or certain segments ofliver or certain types of cells in the liver, such as, e.g.,hepatocytes). In preferred embodiments, a “sample derived from asubject” refers to blood or plasma drawn from the subject. In furtherembodiments, a “sample derived from a subject” refers to liver tissuederived from the subject.

In some embodiments of the methods of the invention, the RNAi agent isadministered to a subject such that the RNAi agent is delivered to aspecific site within the subject. The inhibition of expression ofTMPRSS6 may be assessed using measurements of the level or change in thelevel of TMPRSS6 mRNA or TMPRSS6 protein in a sample derived from fluidor tissue from the specific site within the subject. In preferredembodiments, the site is the liver. The site may also be a subsection orsubgroup of cells from any one of the aforementioned sites. The site mayalso include cells that express a particular type of receptor.

VI. Methods for Treating or Preventing a TMPRSS6 Associated Disorder

The present invention also provides methods for treating or preventingdiseases and conditions that can be modulated by TMPRSS6 geneexpression. For example, the compositions described herein can be usedto treat any disorder associated with iron overload, e.g., a thalassemia(e.g., β-thalassemia or α-thalassemia), primary hemochromatosis,secondary hemochromatosis, severe juvenile hemochromatosis,erythropoietic porphyria, sideroblastic anemia, hemolytic anemia,dyserythropoietic anemia, or sickle-cell anemia. In one embodiment, aTMPRSS6 iRNA is used to treat a hemoglobinopathy. The TMPRSS6 iRNAs ofthe invention can also be used to treat elevated levels of iron due toother conditions, such as chronic alcoholism.

In thalassemias, the bone marrow synthesizes insufficient amounts of ahemoglobin chain; this in turn reduces the production of red blood cellsand causes anemia. Either the α or the β chain may be affected, but βthalassemias are more common. Newborn babies are healthy because theirbodies still produce HbF, which does not have β chains; during the firstfew months of life, the bone marrow switches to producing HbA, andsymptoms start to appear.

β-thalassemias result from mutation with either non-expressing (β°) orlow expressing (β+) alleles of the HBB gene, β-thalassemias vary inseverity depending on the genotype, and include minor/traitβ-thalassemia (β/β° or β/β+), intermedia β-thalassemia (β°/β+), andmajor β-thalassemia (β°/β° or β″7 β+).

Thalassemia intermedia (TI) typically presents with little hemolysis,while major β-thalassemia (TM) is typically accompanied by abundanthemolysis which causes, e.g., anemia and splenomegaly; and highlyineffective erythropoiesis, which causes bone marrow drive (skeletalchanges, oteopenia), increased erythropoietin synthesis,hepato-splenomegaly, consumption of haematinics (megablastic anemia),and high uric acid in blood. The iRNAs of the invention, e.g., TMPRSS6iRNAs, are better suited for treating the iron overload that typicallyaccompanies thalassemia's that are more TI like (e.g., for treatingindividuals having a β°/β+, β/β° or β/β+ genotype).

Symptoms of β-thalassemias also include, e.g., complication due totherapy, e.g., iron overload, which causes endocrinopathy, liverfibrosis and cardiac fibrosis. Administration of an iRNA agent thattargets TMPRSS6 can be effective to treat one or more of these symptoms.

α-thalassemias result from mutation with either non-expressing (α°) orlow expressing (a+) alleles of the HBA1 or HBA2 genes, orthalassemiasvary in severity depending on the genotype, and include traitthalassemia (−α/αα), Hb Bart and Hydrops fetalis (a°/a°), a-Thalaseemiaminor (−/αα), (−α/−α), and HbH disease (−/−a). Lower a-globin chains areproduced, resulting in an excess of β chains in adults and excess γchains in newborns. The excess β chains form unstable tetramers (calledHemoglobin H or HbH of 4 beta chains), which have abnormal oxygendissociation curves. Administration of an iRNA agent that targetsTMPRSS6 can be effective to treat iron overload in a subject who has anα-thalassemias.

Symptoms of hemochromatosis include, e.g., abdominal pain, joint pain,fatigue, lack of energy, weakness, darkening of the skin (often referredto as “bronzing”), and loss of body hair. Administration of an iRNAagent that targets TMPRSS6 can be effective to treat one or more ofthese symptoms.

Other symptoms associated with iron overload include increased risk forliver disease (cirrhosis, cancer), heart attack or heart failure,diabetes mellitus, osteoarthritis, osteoporosis, metabolic syndrome,hypothyroidism, hypogonadism, and in some cases premature death. Ironmismanagement resulting in overload can also accelerate suchneurodegenerative diseases as Alzheimer's, early-onset Parkinson's,Huntington's, epilepsy and multiple sclerosis. Administration of an iRNAagent that targets TMPRSS6, e.g., an iRNA described in Tables 1 or 2 cantreat one or more of these symptoms, or prevent the development orprogression of a disease or disorder that is aggravated by increasediron levels.

The methods of the invention further relate to the use of an iRNA agentor a pharmaceutical composition thereof, e.g., for treating a disorderassociated with iron overload, in combination with other pharmaceuticalsand/or other therapeutic methods, e.g., with known pharmaceuticalsand/or known therapeutic methods, such as, for example, those which arecurrently employed for treating these disorders. For example, in certainembodiments, an iRNA agent targeting TMPRSS6 is administered incombination with, e.g., iron chelators (e.g., desferoxamine), folicacid, a blood transfusion, a phlebotomy, agents to manage ulcers, agentsto increase fetal hemoglobin levels (e.g., hydroxyurea), agents tocontrol infection (e.g., antibiotics and antivirals), agents to treatthrombotic state, or a stem cell or bone marrow transplant. A stem celltransplant can utilize stem cells from an umbilical cord, such as from arelative, e.g., a sibling. Exemplary iron chelators includedesferoxamine, Deferasirox (Exjade), deferiprone, vitamin E, wheat germoil, tocophersolan, and indicaxanthin.

The iRNA agent and an additional therapeutic agent can be administeredin the same composition, e.g., parenterally, or the additionaltherapeutic agent can be administered as part of a separate compositionor by another method described herein. Administration of the iRNA agentand the additional therapeutic agent can be at the same time, or atdifferent times and, in any order.

Administration of the iRNA agent of the invention can lower iron levels,lower ferritin levels, and/or lower transferrin saturation levels. Forexample, administration of the dsRNA can lower serum iron levels and/orlower serum ferritin levels. Transferrin saturation levels can belowered by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, or more. In another embodiment, the transferrinsaturation levels remain lower for 7 days, 10 days, 20 days, 30 days, ormore following administration.

Transferrin saturation levels can be lowered to below 50%, below 45%,below 40%, below 35%, below 35%, below 30%, below 25%, below 20%, below15%, or lower. In another embodiment, the lower transferrin saturationlevels are maintained for 7 days, 10 days, 20 days, 30 days, or morefollowing administration. Transferrin saturation is a measure of theamount of iron bound to serum transferrin, and corresponds to the ratioof serum iron and total iron-binding capacity.

Serum iron levels can be lowered by 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, or more. In another embodiment, the serum iron levels remain lowerfor 7 days, 10 days, 20 days, 30 days, or more following administration.

Administration of the iRNA agent of the invention preferably results inlowered iron levels in the blood, and more particularly in the serum, orin one or more tissues of the mammal. In some embodiments, iron levelsare decreased by at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%,80%, 90% or more, as compared to pretreatment levels.

By “lower” in this context is meant a statistically significant decreasein such level. The decrease can be, for example, at least 10%, at least20%, at least 30%, at least 40% or more, and is preferably down to alevel accepted as within the range of normal for an individual withoutsuch disorder.

Administration of the iRNA agent of the invention can increase serumhepcidin levels, and/or increase hepcidin gene expression. For example,administration of the dsRNA can increase serum hepcidin by at leastabout 10%, 25%, 50%, 100%, 150%, 200%, 250%, 300%, or more. In a furtherexample, administration of the dsRNA can increase hepcidin mRNA levelsby at least about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or greater.

Efficacy of treatment or prevention of disease can be assessed, forexample by measuring disease progression, disease remission, symptomseverity, reduction in pain, quality of life, dose of a medicationrequired to sustain a treatment effect, level of a disease marker or anyother measurable parameter appropriate for a given disease being treatedor targeted for prevention. It is well within the ability of one skilledin the art to monitor efficacy of treatment or prevention by measuringany one of such parameters, or any combination of parameters. Forexample, the levels of transferrin saturation or serum ferritin can bemonitored for efficacy of a given treatment regime.

Iron level tests are typically performed on a sample of a patient'sblood. An iron level test measure the amount of iron in the blood serumthat is being carried by the proteins transferrin. A TIBC (Totaliron-binding capacity) test measures the amount of iron that the bloodwould carry if the transferrin were fully saturated. Since transferrinis produced by the liver, the TIBC can be used to monitor liver functionand nutrition. The transferrin test is a direct measure of transferrin(also called siderophilin) levels in the blood. The saturation level oftransferrin can be calculated by dividing the serum iron level by theTIBC. The ferritin test measures the level of a protein in the bloodthat stores iron for later use by the body.

The iRNA treatments described herein can be used to treat individualsafflicted with a TMPRSS6 associated disorder, e.g., elevated ironlevels, as may be indicated by iron levels in serum e.g., iron levelsmeasuring greater than 350 μg/dL, greater than 500 μg/dL, greater than1000 μg/dL, or more. In an embodiment, elevated levels of iron in serum,e.g., greater than 15, 20, 25, or 30 mg/g dry weight.

The iRNA treatments described herein can also be used to treatindividuals having elevated iron levels, as may be indicated by elevatedferritin levels in serum, e.g., ferritin levels measuring greater than300 μg/L, greater than 500 μg/L, greater than 1000 μg/L, greater than1500 μg/L, greater than 2000 μg/L, greater than 2500 μg/L, or 3000 μg/L,or more.

The iRNA treatments described herein can further be used to treatindividuals having elevated iron levels, as may be indicated by elevatedtransferrin levels in serum, e.g., transferrin levels measuring greaterthan 400 mg/dL, greater than 500 mg/L, greater than 1000 mg/dL, or more.

The iRNA treatments described herein can also be used to treatindividuals having moderately elevated iron levels, as may be indicatedby moderately elevated transferrin saturation levels, e.g., saturationlevels of 40%, 45%, or 50% or more. In addition, the treatment describedherein may also be used to prevent elevated iron levels in individualswith only minor elevations in transferrin saturation. One of skill inthe art can easily monitor the transferrin saturation levels in subjectsreceiving treatment with iRNA as described herein and assay for areduction in transferrin saturation levels of at least 5% or 10%.

The iRNA treatments described herein can be used to treat individualshaving elevated iron levels, as may be indicated by a TIBC value greaterthan 400 μg/dL, greater than 500 μg/dL, or greater than 1000 μg/dL, ormore.

In some embodiments, individuals in need of treatment with an iRNA agentof the invention have decreased hematocrit levels, decreased hemoglobinlevels, increased red blood cell distribution width, increased number ofreticulocytes, decreased number of mature red blood cells, increasedunsaturated iron binding capacity, decreased ineffective erythropoiesis,decreased extradedullary hematopoiesis, and/or decreased HAMP1expression levels.

A patient can be further monitored by assay of blood sugar (glucose)level or a fetoprotein level, by echocardiogram (e.g., to examine theheart's function), electrocardiogram (ECG) (e.g., to look at theelectrical activity of the heart), imaging tests (such as CT scans, MRIand ultrasound), and liver function tests. Excess iron staining or ironconcentrations can be measured on liver biopsy samples, or to confirmthe extent of liver damage, e.g., the stage of liver disease.

A treatment or preventive effect is evident when there is astatistically significant improvement in one or more parameters ofdisease status, or by a failure to worsen or to develop symptoms wherethey would otherwise be anticipated. As an example, a favorable changeof at least 10% in a measurable parameter of disease, and preferably atleast 20%, 30%, 40%, 50% or more can be indicative of effectivetreatment. Efficacy for a given iRNA drug or formulation of that drugcan also be judged using an experimental animal model for the givendisease as known in the art. When using an experimental animal model,efficacy of treatment is evidenced when a statistically significantreduction in a marker or symptom is observed.

Alternatively, the efficacy can be measured by a reduction in theseverity of disease as determined by one skilled in the art of diagnosisbased on a clinically accepted disease severity grading scale.

As used herein, a “subject” includes a human or non-human animal,preferably a vertebrate, and more preferably a mammal. A subject mayinclude a transgenic organism. Most preferably, the subject is a human,such as a human suffering from or predisposed to developing a TMPRSS6associated disorder.

In some embodiments of the methods of the invention, TMPRSS6 expressionis decreased for an extended duration, e.g., at least one week, twoweeks, three weeks, or four weeks or longer. For example, in certaininstances, expression of the TMPRSS6 gene is suppressed by at leastabout 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% by administration of an iRNAagent described herein. In some embodiments, the TMPRSS6 gene issuppressed by at least about 60%, 70%, or 80% by administration of theiRNA agent. In some embodiments, the TMPRSS6 gene is suppressed by atleast about 85%, 90%, or 95% by administration of the double-strandedoligonucleotide. In another embodiment, the TMPRSS6 gene remainssuppressed for 7 days, 10 days, 20 days, 30 days, or more followingadministration.

The RNAi agents of the invention may be administered to a subject usingany mode of administration known in the art, including, but not limitedto subcutaneous, intravenous, intramuscular, intraocular,intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic,cerebrospinal, and any combinations thereof. In preferred embodiments,the agents are administered subcutaneously.

In some embodiments, the administration is via a depot injection. Adepot injection may release the RNAi agent in a consistent way over aprolonged time period. Thus, a depot injection may reduce the frequencyof dosing needed to obtain a desired effect, e.g., a desired inhibitionof TMPRSS6, or a therapeutic or prophylactic effect. A depot injectionmay also provide more consistent serum concentrations. Depot injectionsmay include subcutaneous injections or intramuscular injections. Inpreferred embodiments, the depot injection is a subcutaneous injection.

In some embodiments, the administration is via a pump. The pump may bean external pump or a surgically implanted pump. In certain embodiments,the pump is a subcutaneously implanted osmotic pump. In otherembodiments, the pump is an infusion pump. An infusion pump may be usedfor intravenous, subcutaneous, arterial, or epidural infusions. Inpreferred embodiments, the infusion pump is a subcutaneous infusionpump. In other embodiments, the pump is a surgically implanted pump thatdelivers the RNAi agent to the liver.

Other modes of administration include epidural, intracerebral,intracerebroventricular, nasal administration, intraarterial,intracardiac, intraosseous infusion, intrathecal, and intravitreal, andpulmonary. The mode of administration may be chosen based upon whetherlocal or systemic treatment is desired and based upon the area to betreated. The route and site of administration may be chosen to enhancetargeting.

The method includes administering an iRNA agent, e.g., a dose sufficientto depress levels of TMPRSS6 mRNA for at least 5, more preferably 7, 10,14, 21, 25, 30 or 40 days; and optionally, administering a second singledose of dsRNA, wherein the second single dose is administered at least5, more preferably 7, 10, 14, 21, 25, 30 or 40 days after the firstsingle dose is administered, thereby inhibiting the expression of theTMPRSS6 gene in a subject.

In one embodiment, doses of iRNA agent of the invention are administerednot more than once every four weeks, not more than once every threeweeks, not more than once every two weeks, or not more than once everyweek. In another embodiment, the administrations can be maintained forone, two, three, or six months, or one year or longer. In anotherembodiment, doses of iRNA agent of the invention are administered once aweek for three weeks.

In general, the iRNA agent does not activate the immune system, e.g., itdoes not increase cytokine levels, such as TNF-alpha or IFN-alphalevels. For example, when measured by an assay, such as an in vitro PBMCassay, such as described herein, the increase in levels of TNF-alpha orIFN-alpha, is less than 30%, 20%, or 10% of control cells treated with acontrol dsRNA, such as a dsRNA that does not target TMPRSS6.

For example, a subject can be administered a therapeutic amount of aniRNA agent, such as 0.3 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2.0mg/kg, 2.5 mg/kg, or 3 mg/kg of dsRNA. The iRNA agent can beadministered by intravenous infusion over a period of time, such as overa 5 minute, 10 minute, 15 minute, 20 minute, or 25 minute period. Theadministration is repeated, for example, on a regular basis, such asbiweekly (i.e., every two weeks) for one month, two months, threemonths, four months or longer. After an initial treatment regimen, thetreatments can be administered on a less frequent basis. For example,after administration biweekly for three months, administration can berepeated once per month, for six months or a year or longer.Administration of the iRNA agent can reduce TMPRSS6 levels, e.g., in acell, tissue, blood, urine or other compartment of the patient by atleast 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80% or atleast 90% or more.

Before administration of a full dose of the iRNA agent, patients can beadministered a smaller dose, such as a dose resulting in less than 5%infusion reaction, and monitored for adverse effects, such as anallergic reaction, or for elevated lipid levels or blood pressure. Inanother example, the patient can be monitored for unwantedimmunostimulatory effects, such as increased cytokine (e.g., TNF-alphaor INF-alpha) levels.

Many disorders associated with elevated iron levels are hereditary.Therefore, a patient in need of a TMPRSS6 iRNA may be identified bytaking a family history. A healthcare provider, such as a doctor, nurse,or family member, can take a family history before prescribing oradministering a TMPRSS6 dsRNA. A DNA test may also be performed on thepatient to identify a mutation in the TMPRSS6 gene, before a TMPRSS6dsRNA is administered to the patient. For example, diagnosis ofhereditary hemochromatosis can be confirmed by identifying the two HFE(Hemochromatosis) gene mutations C282Y and H63D, according to GenBankAccession No. CAB07442.1 (GI: 1890180, record dated Oct. 23, 2008).

A treatment or preventive effect is evident when there is astatistically significant improvement in one or more parameters ofdisease status, or by a failure to worsen or to develop symptoms wherethey would otherwise be anticipated. As an example, a favorable changeof at least 10% in a measurable parameter of disease, and preferably atleast 20%, 30%, 40%, 50% or more can be indicative of effectivetreatment. Efficacy for a given iRNA agent of the invention orformulation of that iRNA agent can also be judged using an experimentalanimal model for the given disease as known in the art. When using anexperimental animal model, efficacy of treatment is evidenced when astatistically significant reduction in a marker or symptom is observed.

In one embodiment, the RNAi agent is administered at a dose of betweenabout 0.25 mg/kg to about 50 mg/kg, e.g., between about 0.25 mg/kg toabout 0.5 mg/kg, between about 0.25 mg/kg to about 1 mg/kg, betweenabout 0.25 mg/kg to about 5 mg/kg, between about 0.25 mg/kg to about 10mg/kg, between about 1 mg/kg to about 10 mg/kg, between about 5 mg/kg toabout 15 mg/kg, between about 10 mg/kg to about 20 mg/kg, between about15 mg/kg to about 25 mg/kg, between about 20 mg/kg to about 30 mg/kg,between about 25 mg/kg to about 35 mg/kg, or between about 40 mg/kg toabout 50 mg/kg.

In some embodiments, the RNAi agent is administered at a dose of about0.25 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about8 mg/kg, about 9 mg/kg, about 10 mg/kg, about 11 mg/kg, about 12 mg/kg,about 13 mg/kg, about 14 mg/kg, about 15 mg/kg, about 16 mg/kg, about 17mg/kg, about 18 mg/kg, about 19 mg/kg, about 20 mg/kg, about 21 mg/kg,about 22 mg/kg, about 23 mg/kg, about 24 mg/kg, about 25 mg/kg, about 26mg/kg, about 27 mg/kg, about 28 mg/kg, about 29 mg/kg, 30 mg/kg, about31 mg/kg, about 32 mg/kg, about 33 mg/kg, about 34 mg/kg, about 35mg/kg, about 36 mg/kg, about 37 mg/kg, about 38 mg/kg, about 39 mg/kg,about 40 mg/kg, about 41 mg/kg, about 42 mg/kg, about 43 mg/kg, about 44mg/kg, about 45 mg/kg, about 46 mg/kg, about 47 mg/kg, about 48 mg/kg,about 49 mg/kg or about 50 mg/kg.

In certain embodiments, for example, when a composition of the inventioncomprises a dsRNA as described herein and a lipid, subjects can beadministered a therapeutic amount of iRNA, such as about 0.01 mg/kg toabout 5 mg/kg, about 0.01 mg/kg to about 10 mg/kg, about 0.05 mg/kg toabout 5 mg/kg, about 0.05 mg/kg to about 10 mg/kg, about 0.1 mg/kg toabout 5 mg/kg, about 0.1 mg/kg to about 10 mg/kg, about 0.2 mg/kg toabout 5 mg/kg, about 0.2 mg/kg to about 10 mg/kg, about 0.3 mg/kg toabout 5 mg/kg, about 0.3 mg/kg to about 10 mg/kg, about 0.4 mg/kg toabout 5 mg/kg, about 0.4 mg/kg to about 10 mg/kg, about 0.5 mg/kg toabout 5 mg/kg, about 0.5 mg/kg to about 10 mg/kg, about 1 mg/kg to about5 mg/kg, about 1 mg/kg to about 10 mg/kg, about 1.5 mg/kg to about 5mg/kg, about 1.5 mg/kg to about 10 mg/kg, about 2 mg/kg to about about2.5 mg/kg, about 2 mg/kg to about 10 mg/kg, about 3 mg/kg to about 5mg/kg, about 3 mg/kg to about 10 mg/kg, about 3.5 mg/kg to about 5mg/kg, about 4 mg/kg to about 5 mg/kg, about 4.5 mg/kg to about 5 mg/kg,about 4 mg/kg to about 10 mg/kg, about 4.5 mg/kg to about 10 mg/kg,about 5 mg/kg to about 10 mg/kg, about 5.5 mg/kg to about 10 mg/kg,about 6 mg/kg to about 10 mg/kg, about 6.5 mg/kg to about 10 mg/kg,about 7 mg/kg to about 10 mg/kg, about 7.5 mg/kg to about 10 mg/kg,about 8 mg/kg to about 10 mg/kg, about 8.5 mg/kg to about 10 mg/kg,about 9 mg/kg to about 10 mg/kg, or about 9.5 mg/kg to about 10 mg/kg.Values and ranges intermediate to the recited values are also intendedto be part of this invention. For example, the dsRNA may be administeredat a dose of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1,1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6,2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6,5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1,7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6,8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about10 mg/kg. Values and ranges intermediate to the recited values are alsointended to be part of this invention.

In certain embodiments of the invention, for example, when adouble-stranded RNAi agent includes one or more modifications (e.g.,motifs of three identical modifications on three consecutivenucleotides, including one such motif at or near the cleavage site ofthe agent), six phosphorothioate linkages, and a ligand, such an agentis administered at a dose of about 0.01 to about 0.5 mg/kg, about 0.01to about 0.4 mg/kg, about 0.01 to about 0.3 mg/kg, about 0.01 to about0.2 mg/kg, about 0.01 to about 0.1 mg/kg, about 0.01 mg/kg to about 0.09mg/kg, about 0.01 mg/kg to about 0.08 mg/kg, about 0.01 mg/kg to about0.07 mg/kg, about 0.01 mg/kg to about 0.06 mg/kg, about 0.01 mg/kg toabout 0.05 mg/kg, about 0.02 to about 0.5 mg/kg, about 0.02 to about 0.4mg/kg, about 0.02 to about 0.3 mg/kg, about 0.02 to about 0.2 mg/kg,about 0.02 to about 0.1 mg/kg, about 0.02 mg/kg to about 0.09 mg/kg,about 0.02 mg/kg to about 0.08 mg/kg, about 0.02 mg/kg to about 0.07mg/kg, about 0.02 mg/kg to about 0.06 mg/kg, about 0.02 mg/kg to about0.05 mg/kg, about 0.03 to about 0.5 mg/kg, about 0.03 to about 0.4mg/kg, about 0.03 to about 0.3 mg/kg, about 0.03 to about 0.2 mg/kg,about 0.03 to about 0.1 mg/kg, about 0.03 mg/kg to about 0.09 mg/kg,about 0.03 mg/kg to about 0.08 mg/kg, about 0.03 mg/kg to about 0.07mg/kg, about 0.03 mg/kg to about 0.06 mg/kg, about 0.03 mg/kg to about0.05 mg/kg, about 0.04 to about 0.5 mg/kg, about 0.04 to about 0.4mg/kg, about 0.04 to about 0.3 mg/kg, about 0.04 to about 0.2 mg/kg,about 0.04 to about 0.1 mg/kg, about 0.04 mg/kg to about 0.09 mg/kg,about 0.04 mg/kg to about 0.08 mg/kg, about 0.04 mg/kg to about 0.07mg/kg, about 0.04 mg/kg to about 0.06 mg/kg, about 0.05 to about 0.5mg/kg, about 0.05 to about 0.4 mg/kg, about 0.05 to about 0.3 mg/kg,about 0.05 to about 0.2 mg/kg, about 0.05 to about 0.1 mg/kg, about 0.05mg/kg to about 0.09 mg/kg, about 0.05 mg/kg to about 0.08 mg/kg, orabout 0.05 mg/kg to about 0.07 mg/kg. Values and ranges intermediate tothe foregoing recited values are also intended to be part of thisinvention, e.g., the RNAi agent may be administered to the subject at adose of about 0.015 mg/kg to about 0.45 mg/mg.

For example, the RNAi agent, e.g., RNAi agent in a pharmaceuticalcomposition, may be administered at a dose of about 0.01 mg/kg, 0.0125mg/kg, 0.015 mg/kg, 0.0175 mg/kg, 0.02 mg/kg, 0.0225 mg/kg, 0.025 mg/kg,0.0275 mg/kg, 0.03 mg/kg, 0.0325 mg/kg, 0.035 mg/kg, 0.0375 mg/kg, 0.04mg/kg, 0.0425 mg/kg, 0.045 mg/kg, 0.0475 mg/kg, 0.05 mg/kg, 0.0525mg/kg, 0.055 mg/kg, 0.0575 mg/kg, 0.06 mg/kg, 0.0625 mg/kg, 0.065 mg/kg,0.0675 mg/kg, 0.07 mg/kg, 0.0725 mg/kg, 0.075 mg/kg, 0.0775 mg/kg, 0.08mg/kg, 0.0825 mg/kg, 0.085 mg/kg, 0.0875 mg/kg, 0.09 mg/kg, 0.0925mg/kg, 0.095 mg/kg, 0.0975 mg/kg, 0.1 mg/kg, 0.125 mg/kg, 0.15 mg/kg,0.175 mg/kg, 0.2 mg/kg, 0.225 mg/kg, 0.25 mg/kg, 0.275 mg/kg, 0.3 mg/kg,0.325 mg/kg, 0.35 mg/kg, 0.375 mg/kg, 0.4 mg/kg, 0.425 mg/kg, 0.45mg/kg, 0.475 mg/kg, or about 0.5 mg/kg. Values intermediate to theforegoing recited values are also intended to be part of this invention.

The dose of an RNAi agent that is administered to a subject may betailored to balance the risks and benefits of a particular dose, forexample, to achieve a desired level of TMPRSS6 gene suppression (asassessed, e.g., based on TMPRSS6 mRNA suppression, TMPRSS6 proteinexpression, or a reduction in lipid levels) or a desired therapeutic orprophylactic effect, while at the same time avoiding undesirable sideeffects.

In some embodiments, the RNAi agent is administered in two or moredoses. If desired to facilitate repeated or frequent infusions,implantation of a delivery device, e.g., a pump, semi-permanent stent(e.g., intravenous, intraperitoneal, intracisternal or intracapsular),or reservoir may be advisable. In some embodiments, the number or amountof subsequent doses is dependent on the achievement of a desired effect,e.g., the suppression of a TMPRSS6 gene, or the achievement of atherapeutic or prophylactic effect, e.g., reducing iron overload. Insome embodiments, the RNAi agent is administered according to aschedule. For example, the RNAi agent may be administered once per week,twice per week, three times per week, four times per week, or five timesper week. In some embodiments, the schedule involves regularly spacedadministrations, e.g., hourly, every four hours, every six hours, everyeight hours, every twelve hours, daily, every 2 days, every 3 days,every 4 days, every 5 days, weekly, biweekly, or monthly. In otherembodiments, the schedule involves closely spaced administrationsfollowed by a longer period of time during which the agent is notadministered. For example, the schedule may involve an initial set ofdoses that are administered in a relatively short period of time (e.g.,about every 6 hours, about every 12 hours, about every 24 hours, aboutevery 48 hours, or about every 72 hours) followed by a longer timeperiod (e.g., about 1 week, about 2 weeks, about 3 weeks, about 4 weeks,about 5 weeks, about 6 weeks, about 7 weeks, or about 8 weeks) duringwhich the RNAi agent is not administered. In one embodiment, the RNAiagent is initially administered hourly and is later administered at alonger interval (e.g., daily, weekly, biweekly, or monthly). In anotherembodiment, the RNAi agent is initially administered daily and is lateradministered at a longer interval (e.g., weekly, biweekly, or monthly).In certain embodiments, the longer interval increases over time or isdetermined based on the achievement of a desired effect. In a specificembodiment, the RNAi agent is administered once daily during a firstweek, followed by weekly dosing starting on the eighth day ofadministration. In another specific embodiment, the RNAi agent isadministered every other day during a first week followed by weeklydosing starting on the eighth day of administration.

In some embodiments, the RNAi agent is administered in a dosing regimenthat includes a “loading phase” of closely spaced administrations thatmay be followed by a “maintenance phase”, in which the RNAi agent isadministered at longer spaced intervals. In one embodiment, the loadingphase comprises five daily administrations of the RNAi agent during thefirst week. In another embodiment, the maintenance phase comprises oneor two weekly administrations of the RNAi agent. In a furtherembodiment, the maintenance phase lasts for 5 weeks.

Any of these schedules may optionally be repeated for one or moreiterations. The number of iterations may depend on the achievement of adesired effect, e.g., the suppression of a TMPRSS6 gene, and/or theachievement of a therapeutic or prophylactic effect, e.g., reducing ironlevels or reducing a symptom of thalassemia, e.g., β-thalassemia, orhemotochromatosis.

In another aspect, the invention features, a method of instructing anend user, e.g., a caregiver or a subject, on how to administer an iRNAagent described herein. The method includes, optionally, providing theend user with one or more doses of the iRNA agent, and instructing theend user to administer the iRNA agent on a regimen described herein,thereby instructing the end user.

VII. Kits

The present invention also provides kits for using any of the iRNAagents and/or performing any of the methods of the invention. Such kitsinclude one or more RNAi agent(s) and instructions for use, e.g.,instructions for inhibiting expression of a TMPRSS6 in a cell bycontacting the cell with the RNAi agent(s) in an amount effective toinhibit expression of the TMPRSS6. The kits may optionally furthercomprise means for contacting the cell with the RNAi agent (e.g., aninjection device), or means for measuring the inhibition of TMPRSS6(e.g., means for measuring the inhibition of TMPRSS6 mRNA or TTRprotein). Such means for measuring the inhibition of TMPRSS6 maycomprise a means for obtaining a sample from a subject, such as, e.g., aplasma sample. The kits of the invention may optionally further comprisemeans for administering the RNAi agent(s) to a subject or means fordetermining the therapeutically effective or prophylactically effectiveamount.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the iRNAs and methods featured in the invention,suitable methods and materials are described below. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and notintended to be limiting.

EXAMPLES Materials and Methods

The following materials and methods were used in the Examples.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit(Applied Biosystems, Foster City, Calif., Cat #4368813)

A master mix of 2 μl 10× Buffer, 0.8 μl 25×dNTPs, 2 μl Random primers, 1μl Reverse Transcriptase, 1 μl RNase inhibitor and 3.2 μl of H₂O perreaction was added into 10 μl total RNA. cDNA was generated using aBio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through thefollowing steps: 25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C.hold.

Cell Culture and Transfections

Hep3B cells (ATCC, Manassas, Va.) were grown to near confluence at 37°C. in an atmosphere of 5% CO2 in EMEM (ATCC) supplemented with 10% FBS,streptomycin, and glutamine (ATCC) before being released from the plateby trypsinization. Transfection was carried out by adding 14.8 μl ofOpti-MEM plus 0.2 μl of Lipofectamine RNAiMax per well (Invitrogen,Carlsbad Calif. cat #13778-150) to 5 μl of siRNA duplexes per well intoa 96-well plate and incubated at room temperature for 15 minutes.Subsequently, 80 μl of complete growth media without antibioticcontaining ˜2×10⁴ Hep3B cells were then added to the siRNA mixture.Cells were incubated for 24 hours prior to RNA purification. Single doseexperiments were performed at 10 nM and 0.1 nM final duplexconcentration.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit (Invitrogen, Part#: 610-12)

Cells were harvested and lysed in 150 μl of Lysis/Binding Buffer thenmixed for 5 minute at 850 rpm using a platform shaker (the mixing speedwas the same throughout the process). Ten microliters of magnetic beadsand 80 μl Lysis/Binding Buffer mixture were added to a round bottomplate and mixed for 1 minute. Magnetic beads were captured usingmagnetic stand and the supernatant was removed without disturbing thebeads. After the supernatant was removed, the lysed cells were added tothe remaining beads and mixed for 5 minutes. After the supernatant wasremoved, magnetic beads were washed 2 times with 150 μl Wash Buffer Aand mixed for 1 minute. Beads were capture again and supernatantremoved. Beads were then washed with 150 μl Wash Buffer B, captured andsupernatant was removed. Beads were next washed with 150 μl ElutionBuffer, captured and supernatant removed. Beads were allowed to dry for2 minutes. After drying, 50 μl of Elution Buffer was added and mixed for5 minutes at 75° C. Beads were captured on magnet for 5 minutes, and 50μl of supernatant containing the purified RNA was removed and added to anew 96 well plate.

Real Time PCR

Two al of cDNA was added to a master mix containing 0.5 μl human GAPDHTaqMan Probe (Applied Biosystems Cat #4326317E), 0.5 μl human TMPRSS6TaqMan probe (Applied Biosystems cat # Hs00542184_m1) and 5 μlLightcycler 480 probe master mix (Roche Cat #04887301001) per well in a384 well plate (Roche cat #04887301001). Real time PCR was performed ina Roche LC480 Real Time PCR system (Roche) using the ΔΔCt(RQ) assay.Each duplex was tested in two independent transfections and eachtransfection was assayed in duplicate, unless otherwise noted.

To calculate relative fold change, real time data were analyzed usingthe ΔΔCt method and normalized to assays performed with cellstransfected with 10 nM AD-1955, or mock transfected cells.

The sense and antisense sequences of AD-1955 are: SENSE:5′-cuuAcGcuGAGuAcuucGAdTsdT-3′ (SEQ ID NO: 15); and ANTISENSE:5′-UCGAAGuACUcAGCGuAAGdTsdT-3′ (SEQ ID NO: 16).

TABLE B Abbreviations of nucleotide monomers used in nucleic acidsequence representation. Abbreviation Nucleotide(s) AAdenosine-3′-phosphate Ab beta-L-adenosine-3′-phosphate Af2′-fluoroadenosine-3′-phosphate Afs2′-fluoroadenosine-3′-phosphorothioate As adenosine-3′-phosphorothioateC cytidine-3′-phosphate Cb beta-L-cytidine-3′-phosphate Cf2′-fluorocytidine-3′-phosphate Cfs 2′-fluorocytidine-3′-phosphorothioateCs cytidine-3′-phosphorothioate G guanosine-3′-phosphate Gbbeta-L-guanosine-3′-phosphate Gbs beta-L-guanosine-3′-phosphorothioateGf 2′-fluoroguanosine-3′-phosphate Gfs2′-fluoroguanosine-3′-phosphorothioate Gs guanosine-3′-phosphorothioateT 5′-methyluridine-3′-phosphate Tf2′-fluoro-5-methyluridine-3′-phosphate Tfs2′-fluoro-5-methyluridine-3′-phosphorothioate Ts5-methyluridine-3′-phosphorothioate U Uridine-3′-phosphate Uf2′-fluorouridine-3′-phosphate Ufs 2′-fluorouridine-3′-phosphorothioateUs uridine-3′-phosphorothioate N any nucleotide (G, A, C, T or U) a2′-O-methyladenosine-3′-phosphate as2′-O-methyladenosine-3′-phosphorothioate c2′-O-methylcytidine-3′-phosphate cs2′-O-methylcytidine-3′-phosphorothioate g2′-O-methylguanosine-3′-phosphate gs2′-O-methylguanosine-3′-phosphorothioate t2′-O-methyl-5-methyluridine-3′-phosphate ts2′-O-methyl-5-methyluridine-3′-phosphorothioate u2′-O-methyluridine-3′-phosphate us2′-O-methyluridine-3′-phosphorothioate dT 2′-deoxythymidine dTs2′-deoxythymidine-3′-phosphorothioate dU 2′-deoxyuridine sphosphorothioate linkage L96N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinolHyp-(GalNAc-alkyl)3 (Aeo) 2′-O-methoxyethyladenosine-3′-phosphate (Aeos)2′-O-methoxyethyladenosine-3′-phosphorothioate (Geo)2′-O-methoxyethylguanosine-3′-phosphate (Geos)2′-O-methoxyethylguanosine-3′-phosphorothioate (Teo)2′-O-methoxyethyl-5-methyluridine-3′-phosphate (Teos)2′-O-methoxyethyl-5-methyluridine-3′-phosphorothioate (m5Ceo)2′-O-methoxyethyl-5-methylcytidine-3′-phosphate (m5Ceos)2′-O-methoxyethyl-5-methylcytidine-3′-phosphorothioate (A3m)3′-O-methyladenosine-2′-phosphate (A3mx)3′-O-methyl-xylofuranosyladenosine-2′-phosphate (G3m)3′-O-methylguanosine-2′-phosphate (G3mx)3′-O-methyl-xylofuranosylguanosine-2′-phosphate (C3m)3′-O-methylcytidine-2′-phosphate (C3mx)3′-O-methyl-xylofuranosylcytidine-2′-phosphate (U3m)3′-O-methyluridine-2′-phosphate (U3mx)3′-O-methylxylouridine-2′-phosphate (Chd)2′-O-hexadecyl-cytidine-3′-phosphate (pshe) Hydroxyethylphosphorothioate(Uhd) 2′-O-hexadecyl-uridine-3′-phosphate (Tgn) Thymidine-glycol nucleicacid (GNA) S-Isomer (Cgn) Cytidine-glycol nucleic acid (GNA) (Chd)2′-O-hexadecyl-cytidine-3′-phosphate (Ggn)2′-O-hexadecyl-cytidine-3′-phosphate (Agn) Adenosine-glycol nucleic acid(GNA) P 5′-phosphate (m5Cam)2′-O-(N-methylacetamide)-5-methylcytidine-3′-phosphate (m5Cams)2′-O-(N-methylacetamide)-5-methylcytidine-3′- phosphorothioate (Tam)2′-O-(N-methylacetamide)thymidine-3′-phosphate (Tams)2′-O-(N-methylacetamide)thymidine-3′-phosphorothioate (Aam)2′-O-(N-methylacetamide)adenosine-3′-phosphate (Aams)2′-O-(N-methylacetamide)adenosine-3′-phosphorothioate (Gam)2′-O-(N-methylacetamide)guanosine-3′-phosphate (Gams)2′-O-(N-methylacetamide)guanosine-3′-phosphorothioate Y442-hydroxymethyl-tetrahydrofurane-5-phosphate

Example 1. Design, Specificity and Efficacy Prediction ofOligonucleotides Transcripts

siRNA design was carried out to identify siRNAs targeting human, rhesus(Macaca mulatta), mouse, and rat TMPRSS6 transcripts annotated in theNCBI Gene database (http://www.ncbi.nlm.nih.gov/gene/). Design used thefollowing transcripts from the NCBI RefSeq collection:Human—NM_153609.2; Rhesus—XM_001085203.2 and XM_001085319.1;Mouse—NM_027902.2; Rat—NM_001130556.1. Due to high primate/rodentsequence divergence, siRNA duplexes were designed in several separatebatches, including but not limited to batches containing duplexesmatching human and rhesus transcripts only; human, rhesus, and mousetranscripts only; human, rhesus, mouse, and rat transcripts only; andmouse and rat transcripts only. All siRNA duplexes were designed thatshared 100% identity with the listed human transcript and other speciestranscripts considered in each design batch (above).

The specificity of all possible 19mers was predicted from each sequence.Candidate 19mers that lacked repeats longer than 7 nucleotides were thenselected. These 1259 candidate human/rhesus, 91 human/rhesus/mouse, 37human/rhesus/mouse/rat, and 810 mouse/rat siRNAs were used incomprehensive searches against the appropriate transcriptomes (definedas the set of NM_ and XM_(—) records within the human, rhesus, mouse, orrat NCBI Refseq sets) using an exhaustive “brute-force” algorithmimplemented in the python script ‘BruteForce.py’. The script next parsedthe transcript-oligo alignments to generate a score based on theposition and number of mismatches between the siRNA and any potential‘off-target’ transcript. The off-target score is weighted to emphasizedifferences in the ‘seed’ region of siRNAs, in positions 2-9 from the 5′end of the molecule. Each oligo-transcript pair from the brute-forcesearch was given a mismatch score by summing the individual mismatchscores; mismatches in the position 2-9 were counted as 2.8, mismatchesin the cleavage site positions 10-11 were counted as 1.2, and mismatchesin region 12-19 counted as 1.0. An additional off-target prediction wascarried out by comparing the frequency of heptamers and octomers derivedfrom 3 distinct, seed-derived hexamers of each oligo. The hexamers frompositions 2-7 relative to the 5′ start were used to create 2 heptamersand one octomer. Heptamer1 was created by adding a 3′ A to the hexamer;heptamer2 was created by adding a 5′ A to the hexamer; the octomer wascreated by adding an A to both 5′ and 3′ ends of the hexamer. Thefrequency of octomers and heptamers in the human, rhesus, mouse, or rat3′UTRome (defined as the subsequence of the transcriptome from NCBI'sRefseq database where the end of the coding region, the ‘CDS’, isclearly defined) was pre-calculated. The octomer frequency wasnormalized to the heptamer frequency using the median value from therange of octomer frequencies. A ‘mirSeedScore’ was then calculated bycalculating the sum of ((3×normalized octomer count)+(2×heptamer2count)+(1×heptamer1 count)).

Both siRNA strands were assigned to a category of specificity accordingto the calculated scores: a score above 3 qualified as highly specific,equal to 3 as specific and between 2.2 and 2.8 qualified as moderatelyspecific. The siRNAs were sorted by the specificity of the antisensestrand. Duplexes from the human/rhesus and mouse/rat sets whoseantisense oligos lacked GC at the first position, lacked G at bothpositions 13 and 14, and had 3 or more Us or As in the seed region(characteristics of duplexes with high predicted efficacy) were thenselected. Similarly, duplexes from the human/rhesus/mouse andhuman/rhesus/mouse/rat sets that had had 3 or more Us or As in the seedregion were selected.

Candidate GalNAc-conjugated duplexes, 21 and 23 nucleotides long on thesense and antisense strands respectively, were designed by extendingantisense 19mers 4 additional nucleotides in the 3′ direction(preserving perfect complementarity with the target transcript). Thesense strand was specified as the reverse complement of the first 21nucleotides of the antisense 23mer. Duplexes were selected thatmaintained perfect matches to all selected species transcripts acrossall 23 nucleotides.

siRNA Sequence Selection

A total of 39 sense and 39 antisense derived human/rhesus, 6 sense and 6antisense derived human/rhesus/mouse, 3 sense and 3 antisense derivedhuman/rhesus/mouse/rat, and 16 sense and 16 antisense derived mouse/ratsiRNA 21/23mer oligos were synthesized and formed into GalNAc-conjugatedduplexes.

The sequences of the sense and antisense strands of the modifiedduplexes are shown in Table 1, and the sequences of the sense andantisense strands of the unmodified duplexes are shown in Table 2.

TABLE 1 TMPRSS6 modified sequences Sense SEQ SEQ Duplex sequence IDAntisense ID ID ID Sense sequence NO: sequence Antisense sequence NO:AD- A-119159.1 UfsgsGfcCfuGfgAfGfAfgGfuGfuCfcUfuCfL96 17 A-119160.1usUfsgAfaGfgAfcAfccuCfuCfcAfgGfcsCfsa  65 58686.1 AD- A-119175.1GfsgsGfgUfgCfuAfCfUfcUfgGfuAfuUfuCfL96 18 A-119176.1asGfsgAfaAfuAfcCfagaGfuAfgCfaCfcsCfsc  66 58687.1 AD- A-119191.1CfsasAfcGfgCfcUfGfGfaUfgAfgAfgAfaAfL96 19 A-119192.1asGfsuUfuCfuCfuCfaucCfaGfgCfcGfusUfsg  67 58688.1 AD- A-119207.1AfsusCfgCfcAfcUfUfCfuCfcCfaGfgAfuCfL96 20 A-119208.1asAfsgAfuCfcUfgGfgagAfaGfuGfgCfgsAfsu  68 58689.1 AD- A-119223.1GfsgsUfgGfcAfgGfAfGfgUfgGfcAfuCfuUfL96 21 A-119224.1asCfsaAfgAfuGfcCfaccUfcCfuGfcCfasCfsc  69 58690.1 AD- A-119161.1GfsasCfcGfaCfuGfGfCfcAfuGfuAfuGfaCfL96 22 A-119162.1asCfsgUfcAfuAfcAfuggCfcAfgUfcGfgsUfsc  70 58692.1 AD- A-119177.1GfsgsUfgUfgCfgGfGfUfgCfaCfuAfuGfgCfL96 23 A-119178.1asAfsgCfcAfuAfgUfgcaCfcCfgCfaCfasCfsc  71 58693.1 AD- A-119193.1GfsgsCfcUfgGfaUfGfAfgAfgAfaAfcUfgCfL96 24 A-119194.1asCfsgCfaGfuUfuCfucuCfaUfcCfaGfgsCfsc  72 58694.1 AD- A-119209.1CfsusCfuGfgUfaUfUfUfcCfuAfgGfgUfaCfL96 25 A-119210.1usUfsgUfaCfcCfuAfggaAfaUfaCfcAfgsAfsg  73 58695.1 AD- A-119225.1GfscsCfcCfuGfgUfCfUfaAfcUfuGfgGfaUfL96 26 A-119226.1asGfsaUfcCfcAfaGfuuaGfaCfcAfgGfgsGfsc  74 58696.1 AD- A-119163.1GfsasGfgCfaGfaAfGfUfaUfgAfuUfuGfcCfL96 27 A-119164.1asCfsgGfcAfaAfuCfauaCfuUfcUfgCfcsUfsc  75 58698.1 AD- A-119179.1AfsasGfcCfaGfuGfUfGfaAfaGfaCfaUfaGfL96 28 A-119180.1asGfscUfaUfgUfcUfuucAfcAfcUfgGfcsUfsu  76 58699.1 AD- A-119195.1GfscsCfgGfgAfcCfGfAfcUfgGfcCfaUfgUfL96 29 A-119196.1asUfsaCfaUfgGfcCfaguCfgGfuCfcCfgsGfsc  77 58700.1 AD- A-119211.1CfsusCfcAfgGfuUfCfGfgGfgUfcGfaCfaCfL96 30 A-119212.1asUfsgUfgUfcGfaCfcccGfaAfcCfuGfgsAfsg  78 58701.1 AD- A-119227.1AfsgsCfcCfcUfgGfUfCfuAfaCfuUfgGfgAfL96 31 A-119228.1gsAfsuCfcCfaAfgUfuagAfcCfaGfgGfgsCfsu  79 58702.1 AD- A-119165.1UfscsGfcCfaCfuUfCfUfcCfcAfgGfaUfcUfL96 32 A-119166.1usAfsaGfaUfcCfuGfggaGfaAfgUfgGfcsGfsa  80 58704.1 AD- A-119181.1AfscsUfcUfgGfuAfUfUfuCfcUfaGfgGfuAfL96 33 A-119182.1usGfsuAfcCfcUfaGfgaaAfuAfcCfaGfasGfsu  81 58705.1 AD- A-119197.1UfscsGfcUfgAfcCfGfCfuGfgGfuGfaUfaAfL96 34 A-119198.1usGfsuUfaUfcAfcCfcagCfgGfuCfaGfcsGfsa  82 58706.1 AD- A-119213.1GfscsCfcCfaAfcGfGfCfcUfgGfaUfgAfgAfL96 35 A-119214.1usCfsuCfuCfaUfcCfaggCfcGfuUfgGfgsGfsc  83 58707.1 AD- A-119229.1GfscsCfaAfgCfaGfGfGfgGfaCfaAfgUfaUfL96 36 A-119230.1gsAfsaUfaCfuUfgUfcccCfcUfgCfuUfgsGfsc  84 58708.1 AD- A-119167.1UfscsCfcCfuAfcAfGfGfgCfcGfaGfuAfcGfL96 37 A-119168.1usUfscGfuAfcUfcGfgccCfuGfuAfgGfgsGfsa  85 58710.1 AD- A-119183.1CfsusGfgGfuUfgUfUfAfcCfgCfuAfcAfgCfL96 38 A-119184.1usAfsgCfuGfuAfgCfgguAfaCfaAfcCfcsAfsg  86 58711.1 AD- A-119199.1CfsusGfgCfcUfgGfAfGfaGfgUfgUfcCfuUfL96 39 A-119200.1usGfsaAfgGfaCfaCfcucUfcCfaGfgCfcsAfsg  87 58712.1 AD- A-119215.1GfsusGfcGfgGfuGfCfAfcUfaUfgGfcUfuGfL96 40 A-119216.1usAfscAfaGfcCfaUfaguGfcAfcCfcGfcsAfsc  88 58713.1 AD- A-119231.1UfsgsGfcAfgGfaGfGfUfgGfcAfuCfuUfgUfL96 41 A-119232.1asGfsaCfaAfgAfuGfccaCfcUfcCfuGfcsCfsa  89 58714.1 AD- A-119169.1CfscsCfuAfcAfgGfGfCfcGfaGfuAfcGfaAfL96 42 A-119170.1asCfsuUfcGfuAfcUfcggCfcCfuGfuAfgsGfsg  90 58716.1 AD- A-119185.1AfscsCfuGfcUfuCfUfUfcUfgGfuUfcAfuUfL96 43 A-119186.1asGfsaAfuGfaAfcCfagaAfgAfaGfcAfgsGfsu  91 58717.1 AD- A-119201.1UfsgsCfcUfgUfgAfUfGfgGfgUfcAfaGfgAfL96 44 A-119202.1asGfsuCfcUfuGfaCfcccAfuCfaCfaGfgsCfsa  92 58718.1 AD- A-119217.1CfsasGfcUfuCfgGfAfAfgCfcCfcUfgGfuCfL96 45 A-119218.1usAfsgAfcCfaGfgGfgcuUfcCfgAfaGfcsUfsg  93 58719.1 AD- A-119233.1CfscsCfcUfgGfuCfUfAfaCfuUfgGfgAfuCfL96 46 A-119234.1csAfsgAfuCfcCfaAfguuAfgAfcCfaGfgsGfsg  94 58720.1 AD- A-119171.1UfsgsCfuUfcUfuCfUfGfgUfuCfaUfuCfuCfL96 47 A-119172.1usGfsgAfgAfaUfgAfaccAfgAfaGfaAfgsCfsa  95 58721.1 AD- A-119187.1CfscsCfaAfcGfgCfCfUfgGfaUfgAfgAfgAfL96 48 A-119188.1usUfsuCfuCfuCfaUfccaGfgCfcGfuUfgsGfsg  96 58722.1 AD- A-119203.1AfsasGfgGfcCfuGfCfAfcAfgCfuAfcUfaCfL96 49 A-119204.1usCfsgUfaGfuAfgCfuguGfcAfgGfcCfcsUfsu  97 58723.1 AD- A-119219.1GfsusCfuAfaCfuUfGfGfgAfuCfuGfgGfaAfL96 50 A-119220.1csAfsuUfcCfcAfgAfuccCfaAfgUfuAfgsAfsc  98 58724.1 AD- A-119235.1AfsgsCfuUfcGfgAfAfGfcCfcCfuGfgUfcUfL96 51 A-119236.1usUfsaGfaCfcAfgGfggcUfuCfcGfaAfgsCfsu  99 58725.1 AD- A-119173.1CfscsAfgUfgUfgAfAfAfgAfcAfuAfgCfuGfL96 52 A-119174.1usGfscAfgCfuAfuGfucuUfuCfaCfaCfusGfsg 100 58726.1 AD- A-119189.1CfscsAfgGfuUfcGfGfGfgUfcGfaCfaCfaUfL96 53 A-119190.1asGfsaUfgUfgUfcGfaccCfcGfaAfcCfusGfsg 101 58727.1 AD- A-119205.1UfscsCfaCfgCfuGfGfGfuUfgUfuAfcCfgCfL96 54 A-119206.1usAfsgCfgGfuAfaCfaacCfcAfgCfgUfgsGfsa 102 58728.1 AD- A-119221.1UfsgsCfcAfaGfcAfGfGfgGfgAfcAfaGfuAfL96 55 A-119222.1asAfsuAfcUfuGfuCfcccCfuGfcUfuGfgsCfsa 103 58729.1 AD- A-119241.1AfsusCfcAfgAfaCfAfGfgAfgGfcUfgUfgUfL96 56 A-119242.1csCfsaCfaCfaGfcCfuccUfgUfuCfuGfgsAfsu 104 58697.1 AD- A-119243.1UfsusCfaCfcUfcCfCfAfgAfuCfuCfcCfuCfL96 57 A-119244.1gsUfsgAfgGfgAfgAfucuGfgGfaGfgUfgsAfsa 105 58703.1 AD- A-119245.1CfscsUfcCfgAfgGfGfUfgAfgUfgGfcCfaUfL96 58 A-119246.1csCfsaUfgGfcCfaCfucaCfcCfuCfgGfasGfsg 106 58709.1 AD- A-119247.1UfscsCfaGfaAfcAfGfGfaGfgCfuGfuGfuGfL96 59 A-119248.1gsCfscAfcAfcAfgCfcucCfuGfuUfcUfgsGfsa 107 58715.1 AD- A-119237.1GfsusGfuCfcUfcCfGfAfgGfgUfgAfgUfgGfL96 60 A-119238.1gsGfscCfaCfuCfaCfccuCfgGfaGfgAfcsAfsc 108 58730.1 AD- A-119249.1UfsusCfgGfgGfuCfGfAfcAfcAfuCfuGfuGfL96 61 A-119250.1csCfscAfcAfgAfuGfuguCfgAfcCfcCfgsAfsa 109 58731.1 AD- A-119251.1UfscsGfgGfgUfcGfAfCfaCfaUfcUfgUfgGfL96 62 A-119252.1csCfscCfaCfaGfaUfgugUfcGfaCfcCfcsGfsa 110 58734.1 AD- A-119253.1UfsgsCfuUfcCfaGfGfAfgGfaCfaGfcAfuGfL96 63 A-119254.1gsCfscAfuGfcUfgUfccuCfcUfgGfaAfgsCfsa 111 58737.1 AD- A-120243.1UfscsUfgGfuAfuUfUfCfcUfaGfgGfuAfcAfL96 64 A-120244.1usGfsuAfcCfcUfaGfgaaAfuAfcCfaGfasgsu 112 59743.1

TABLE 2 TMPRSS6 unmodified sequences Sense SEQ SEQ Duplex sequence IDPosition in Antisense ID Position in ID ID Sense sequence NO:NM_153609.2 sequence Antisense sequence NO: NM_153609.2 AD- A-UGGCCUGGAGAGGUGUCCUUC 113 2041-2063 A- UUGAAGGACACCUCUCCAGGCCA 1612041-2063 58686.1 119159.1 119160.1 AD- A- GGGGUGCUACUCUGGUAUUUC 114 319-341 A- AGGAAAUACCAGAGUAGCACCCC 162  319-341 58687.1 119175.1119176.1 AD- A- CAACGGCCUGGAUGAGAGAAA 115 1557-1579 A-AGUUUCUCUCAUCCAGGCCGUUG 163 1557-1579 58688.1 119191.1 119192.1 AD- A-AUCGCCACUUCUCCCAGGAUC 116  401-423 A- AAGAUCCUGGGAGAAGUGGCGAU 164 401-423 58689.1 119207.1 119208.1 AD- A- GGUGGCAGGAGGUGGCAUCUU 1172665-2688 A- ACAAGAUGCCACCUCCUGCCACC 165 2665-2688 58690.1 119223.1119224.1 AD- A- GACCGACUGGCCAUGUAUGAC 118  922-944 A-ACGUCAUACAUGGCCAGUCGGUC 166  922-944 58692.1 119161.1 119162.1 AD- A-GGUGUGCGGGUGCACUAUGGC 119 1444-1466 A- AAGCCAUAGUGCACCCGCACACC 1671444-1466 58693.1 119177.1 119178.1 AD- A- GGCCUGGAUGAGAGAAACUGC 1201561-1583 A- ACGCAGUUUCUCUCAUCCAGGCC 168 1561-1583 58694.1 119193.1119194.1 AD- A- CUCUGGUAUUUCCUAGGGUAC 121  328-350 A-UUGUACCCUAGGAAAUACCAGAG 169  328-350 58695.1 119209.1 119210.1 AD- A-GCCCCUGGUCUAACUUGGGAU 122 2966-2989 A- AGAUCCCAAGUUAGACCAGGGGC 1702966-2989 58696.1 119225.1 119226.1 AD- A- GAGGCAGAAGUAUGAUUUGCC 1231281-1303 A- ACGGCAAAUCAUACUUCUGCCUC 171 1281-1303 58698.1 119163.1119164.1 AD- A- AAGCCAGUGUGAAAGACAUAG 124  731-753 A-AGCUAUGUCUUUCACACUGGCUU 172  731-753 58699.1 119179.1 119180.1 AD- A-GCCGGGACCGACUGGCCAUGU 125  917-939 A- AUACAUGGCCAGUCGGUCCCGGC 173 917-939 58700.1 119195.1 119196.1 AD- A- CUCCAGGUUCGGGGUCGACAC 1261894-1916 A- AUGUGUCGACCCCGAACCUGGAG 174 1894-1916 58701.1 119211.1119212.1 AD- A- AGCCCCUGGUCUAACUUGGGA 127 2965-2988 A-GAUCCCAAGUUAGACCAGGGGCU 175 2965-2988 58702.1 119227.1 119228.1 AD- A-UCGCCACUUCUCCCAGGAUCU 128  402-424 A- UAAGAUCCUGGGAGAAGUGGCGA 176 402-424 58704.1 119165.1 119166.1 AD- A- ACUCUGGUAUUUCCUAGGGUA 129 327-349 A- UGUACCCUAGGAAAUACCAGAGU 177  327-349 58705.1 119181.1119182.1 AD- A- UCGCUGACCGCUGGGUGAUAA 130 1934-1956 A-UGUUAUCACCCAGCGGUCAGCGA 178 1934-1956 58706.1 119197.1 119198.1 AD- A-GCCCCAACGGCCUGGAUGAGA 131 1553-1575 A- UCUCUCAUCCAGGCCGUUGGGGC 1791553-1575 58707.1 119213.1 119214.1 AD- A- GCCAAGCAGGGGGACAAGUAU 1322610-2633 A- GAAUACUUGUCCCCCUGCUUGGC 180 2610-2633 58708.1 119229.1119230.1 AD- A- UCCCCUACAGGGCCGAGUACG 133  680-702 A-UUCGUACUCGGCCCUGUAGGGGA 181  680-702 58710.1 119167.1 119168.1 AD- A-CUGGGUUGUUACCGCUACAGC 134  769-791 A- UAGCUGUAGCGGUAACAACCCAG 182 769-791 58711.1 119183.1 119184.1 AD- A- CUGGCCUGGAGAGGUGUCCUU 1352040-2062 A- UGAAGGACACCUCUCCAGGCCAG 183 2040-2062 58712.1 119199.1119200.1 AD- A- GUGCGGGUGCACUAUGGCUUG 136 1447-1469 A-UACAAGCCAUAGUGCACCCGCAC 184 1447-1469 58713.1 119215.1 119216.1 AD- A-UGGCAGGAGGUGGCAUCUUGU 137 2667-2690 A- AGACAAGAUGCCACCUCCUGCCA 1852667-2690 58714.1 119231.1 119232.1 AD- A- CCCUACAGGGCCGAGUACGAA 138 682-704 A- ACUUCGUACUCGGCCCUGUAGGG 186  682-704 58716.1 119169.1119170.1 AD- A- ACCUGCUUCUUCUGGUUCAUU 139  559-581 A-AGAAUGAACCAGAAGAAGCAGGU 187  559-581 58717.1 119185.1 119186.1 AD- A-UGCCUGUGAUGGGGUCAAGGA 140 1530-1552 A- AGUCCUUGACCCCAUCACAGGCA 1881530-1552 58718.1 119201.1 119202.1 AD- A- CAGCUUCGGAAGCCCCUGGUC 1412955-2978 A- UAGACCAGGGGCUUCCGAAGCUG 189 2955-2978 58719.1 119217.1119218.1 AD- A- CCCCUGGUCUAACUUGGGAUC 142 2967-2990 A-CAGAUCCCAAGUUAGACCAGGGG 190 2967-2990 58720.1 119233.1 119234.1 AD- A-UGCUUCUUCUGGUUCAUUCUC 143  562-584 A- UGGAGAAUGAACCAGAAGAAGCA 191 562-584 58721.1 119171.1 119172.1 AD- A- CCCAACGGCCUGGAUGAGAGA 1441555-1577 A- UUUCUCUCAUCCAGGCCGUUGGG 192 1555-1577 58722.1 119187.1119188.1 AD- A- AAGGGCCUGCACAGCUACUAC 145 1054-1076 A-UCGUAGUAGCUGUGCAGGCCCUU 193 1054-1076 58723.1 119203.1 119204.1 AD- A-GUCUAACUUGGGAUCUGGGAA 146 2973-2996 A- CAUUCCCAGAUCCCAAGUUAGAC 1942973-2996 58724.1 119219.1 119220.1 AD- A- AGCUUCGGAAGCCCCUGGUCU 1472956-2979 A- UUAGACCAGGGGCUUCCGAAGCU 195 2956-2979 58725.1 119235.1119236.1 AD- A- CCAGUGUGAAAGACAUAGCUG 148  734-756 A-UGCAGCUAUGUCUUUCACACUGG 196  734-756 58726.1 119173.1 119174.1 AD- A-CCAGGUUCGGGGUCGACACAU 149 1896-1918 A- AGAUGUGUCGACCCCGAACCUGG 1971896-1918 58727.1 119189.1 119190.1 AD- A- UCCACGCUGGGUUGUUACCGC 150 763-785 A- UAGCGGUAACAACCCAGCGUGGA 198  763-785 58728.1 119205.1119206.1 AD- A- UGCCAAGCAGGGGGACAAGUA 151 2609-2632 A-AAUACUUGUCCCCCUGCUUGGCA 199 2609-2632 58729.1 119221.1 119222.1 AD- A-AUCCAGAACAGGAGGCUGUGU 152 1324-1346 A- CCACACAGCCUCCUGUUCUGGAU 2001324-1346 58697.1 119241.1 119242.1 AD- A- UUCACCUCCCAGAUCUCCCUC 1531414-1436 A- GUGAGGGAGAUCUGGGAGGUGAA 201 1414-1436 58703.1 119243.1119244.1 AD- A- CCUCCGAGGGUGAGUGGCCAU 154 1862-1884 A-CCAUGGCCACUCACCCUCGGAGG 202 1862-1884 58709.1 119245.1 119246.1 AD- A-UCCAGAACAGGAGGCUGUGUG 155 1325-1347 A- GCCACACAGCCUCCUGUUCUGGA 2031325-1347 58715.1 119247.1 119248.1 AD- A- GUGUCCUCCGAGGGUGAGUGG 1561858-1880 A- GGCCACUCACCCUCGGAGGACAC 204 1858-1880 58730.1 119237.1119238.1 AD- A- UUCGGGGUCGACACAUCUGUG 157 1901-1923 A-CCCACAGAUGUGUCGACCCCGAA 205 1901-1923 58731.1 119249.1 119250.1 AD- A-UCGGGGUCGACACAUCUGUGG 158 1902-1924 A- CCCCACAGAUGUGUCGACCCCGA 2061902-1924 58734.1 119251.1 119252.1 AD- A- UGCUUCCAGGAGGACAGCAUG 1591966-1988 A- GCCAUGCUGUCCUCCUGGAAGCA 207 1966-1988 58737.1 119253.1119254.1 AD- A- UCUGGUAUUUCCUAGGGUACA 160 A- UGUACCCUAGGAAAUACCAGAGU 20859743.1 120243.1 120244.1

Example 2. In Vitro Single Dose Screen

The modified and conjugated TMPRSS6 siRNA duplexes were also evaluatedfor efficacy by transfection assays in human cell line Hep3B. TMPRSS6siRNAs were transfected at two doses, 10 nM and 0.1 nM. The results ofthese assays are shown in Table 3 and the data are expressed as afraction of the message remaining in cells transfected with siRNAstargeting TMPRSS6, relative to cells transfected with a negative controlsiRNA, AD-1955±the standard deviation (SD).

TABLE 3 TMPRSS6 single dose screen. Duplex ID Avg 10 nM SD 10 nM Avg 0.1nM SD 0.1 nM AD-58686.1 71.58 18.94 103.29 32.00 AD-58687.1 89.33 13.14104.94 20.06 AD-58688.1 34.16 11.36 87.18 8.43 AD-58689.1 79.82 7.28110.37 6.08 AD-58690.1 69.10 9.83 99.92 24.84 AD-58692.1 79.21 5.67136.49 0.84 AD-58693.1 77.29 12.12 106.01 17.97 AD-58694.1 50.51 10.3689.47 3.84 AD-58695.1 54.37 5.75 87.66 13.59 AD-58696.1 93.26 0.06 84.793.84 AD-58697.1 72.95 23.41 98.98 10.29 AD-58698.1 42.61 7.81 109.9816.78 AD-58699.1 24.93 8.58 79.71 12.55 AD-58700.1 74.10 15.37 89.757.80 AD-58701.1 79.18 8.18 89.70 9.98 AD-58702.1 96.43 18.38 113.0510.65 AD-58703.1 79.15 28.50 97.30 6.79 AD-58704.1 67.92 0.87 92.26 1.24AD-58705.1 59.50 20.47 99.25 3.28 AD-58706.1 71.67 0.75 102.38 14.88AD-58707.1 77.89 22.26 97.52 1.31 AD-58708.1 73.87 9.61 98.38 1.81AD-58709.1 94.62 4.69 100.73 16.10 AD-58710.1 59.19 10.57 95.23 11.99AD-58711.1 63.62 16.83 103.11 3.66 AD-58712.1 65.79 6.96 81.58 1.50AD-58713.1 84.14 26.41 101.56 5.60 AD-58714.1 64.73 6.06 102.37 1.63AD-58715.1 91.05 18.67 101.08 11.00 AD-58716.1 70.07 13.02 97.20 2.98AD-58717.1 11.27 6.91 66.56 4.32 AD-58718.1 62.10 18.62 89.01 15.30AD-58719.1 72.94 18.26 91.58 9.97 AD-58720.1 60.51 14.43 90.92 5.68AD-58721.1 17.72 7.70 56.72 2.57 AD-58722.1 51.65 11.33 81.44 0.50AD-58723.1 53.27 21.60 94.25 16.20 AD-58724.1 58.03 49.89 77.11 4.63AD-58725.1 54.58 40.10 76.12 1.59 AD-58726.1 10.33 9.88 42.75 7.97AD-58727.1 62.80 26.45 83.23 13.10 AD-58728.1 49.36 36.27 83.30 1.74AD-58729.1 43.83 61.99 73.54 19.33 AD-58730.1 59.60 41.85 76.12 1.03AD-58731.1 85.29 24.78 128.06 32.14 AD-58734.1 85.71 10.74 101.75 6.11AD-58737.1 79.87 10.59 114.89 7.46

Example 3. In Vivo Single Dose Screen Using AD-59743

The ability of AD-59743 to suppress expression of TMPRSS6 protein wasassessed by measuring levels of TMPRSS6 and hepcidin mRNA in the liverof wild-type C57BL/6 mice following administration of AD-59743. A singledose of 1, 3 or 10 mg/kg of AD-59743 was administered subcutaneously,and the mice were sacrificed on day 3 or day 7. Levels of TMPRSS6 andhepcidin mRNA in the liver were measured by qPCR using the methodsdescribed above. A control group received injections with PBS.

The levels of TMPRSS6 mRNA following administration of AD-59743 areshown in FIG. 1, and the levels of hepcidin mRNA followingadministration of AD-59743 are shown in FIG. 2. The results demonstratea dose-dependent decrease in the levels of TMPRSS6 transcripts that issustained through day 7.

Example 4. In Vivo Effect of TMPRSS6 iRNA Agents in Combination with anIron Chelator

The purpose of this study was to test the effect of co-administeredTMPRSS6 specific siRNA and iron chelators on iron levels. In the study,6-week old wild-type C57BL/6 and thalassemic Th3/+ mice (Douet et al.,Am. J. Pathol. (2011), 178(2):774-83) were fed low-iron diets containing3-5 ppm iron. The mice were administered intravenously the formulationAF-011-46273 containing deferiprone, an iron chelator at a dose of 250mg/kg/day and an iRNA agent with the following structure:oligoSeq-sense—uGGuAuuuccuAGGGuAcAdTsdT (SEQ ID NO: 209);oligoSeq-antisense—UGuACCCuAGGAAAuACcAdTsdT (SEQ ID NO: 210). Theformulation also contained MC-3/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5.Liver and spleen tissues were collected and tissue nonheme ironconcentrations were determined as described previously (see, e.g.,Schmidt et al. (2013) Blood 121(7):1200-8; Cook, J D, et al. Tissue ironstores. In: Cook J D, editor. Methods in Hematology. Vol 1. New York,N.Y.: Churchill Livingstone Press; 1980. p. 104-109).

The results of these experiments demonstrate an additive effect ofAD-46273 and deferiprone in Th3/+ mice, with the decreased iron levelsrelative to the negative controls.

Example 5. Design, Specificity and Efficacy Prediction ofOligonucleotides Transcripts

siRNA design was carried out to identify siRNAs targeting human,cynomolgus monkey (Macaca fascicularis; henceforth “cyno”), mouse, andrat TMPRSS6 transcripts annotated in the NCBI Gene database(http://www.ncbi.nlm.nih.gov/gene/). Design used the followingtranscripts from the NCBI RefSeq collection: Human—NM_153609.2;Mouse—NM_027902.2; Rat—NM_001130556.1. For cyno, a transcript sequencewas obtained via alignment with human TMPRSS6 of sequence assembled fromtwo accessions: “ENSP00000384964 [mRNA] locus=chr10:82446450:82485403:-”and FR874253.1, available from the M. fascicularis genome project andNCBI Nucleotide databases, respectively(http://macaque.genomics.org.cn/page/species/download.jsp andhttp://www.ncbi.nlm.nih.gov/nucleotide/). Due to high primate/rodentsequence divergence, siRNA duplexes were designed in several separatebatches, including but not limited to batches containing duplexesmatching human and cyno transcripts only; human, cyno, and mousetranscripts only; and human, cyno, mouse, and rat transcripts only. MostsiRNA duplexes were designed that shared 100% identity in the designatedregion with the listed human transcript and other species transcriptsconsidered in each design batch (above). In some instances, mismatchesbetween duplex and mRNA target were allowed at the first antisense (lastsense) position when the antisense strand:target mRNA complementarybasepair was a GC or CG pair. In these cases, duplexes were designedwith UA or AU pairs at the first antisense:last sense pair. Thus theduplexes maintained complementarity but were mismatched with respect totarget (U:C, U:G, A:C, or A:G).

The specificity of all possible 19mers was predicted from each sequence.Candidate 19mers that lacked repeats longer than 7 nucleotides were thenselected. These 1128 candidate human/cyno, 69 human/cyno/mouse, and 23human/cyno/mouse/rat siRNAs were used in comprehensive searches againstthe appropriate transcriptomes (defined as the set of NM_ and XM_records within the human, mouse, or rat NCBI Refseq sets, and the cynotranscriptome set in NCBI nucleotide) using an exhaustive “brute-force”algorithm implemented in the python script ‘BruteForce.py’. The scriptnext parsed the transcript-oligo alignments to generate a score based onthe position and number of mismatches between the siRNA and anypotential ‘off-target’ transcript. The off-target score is weighted toemphasize differences in the ‘seed’ region of siRNAs, in positions 2-9from the 5′ end of the molecule. Each oligo-transcript pair from thebrute-force search was given a mismatch score by summing the individualmismatch scores; mismatches in the position 2-9 were counted as 2.8,mismatches in the cleavage site positions 10-11 were counted as 1.2, andmismatches in region 12-19 counted as 1.0. An additional off-targetprediction was carried out by comparing the frequency of heptamers andoctomers derived from 3 distinct, seed-derived hexamers of each oligo.The hexamers from positions 2-7 relative to the 5′ start were used tocreate 2 heptamers and one octomer. Heptamer1 was created by adding a 3′A to the hexamer; heptamer2 was created by adding a 5′ A to the hexamer;the octomer was created by adding an A to both 5′ and 3′ ends of thehexamer. The frequency of octomers and heptamers in the human, cyno,mouse, or rat 3′UTRome (defined as the subsequence of the transcriptomefrom NCBI's Refseq database where the end of the coding region, the‘CDS’, is clearly defined) was pre-calculated. The octomer frequency wasnormalized to the heptamer frequency using the median value from therange of octomer frequencies. A ‘mirSeedScore’ was then calculated bycalculating the sum of ((3×normalized octomer count)+(2×heptamer2count)+(1×heptamer1 count)).

Both siRNAs strands were assigned to a category of specificity accordingto the calculated scores: a score above 3 qualifies as highly specific,equal to 3 as specific and between 2 and 2.8 as moderately specific. Wesorted by the specificity of the antisense strand. We then selectedmoderately (or higher) specific duplexes whose antisense oligospossessed characteristics of duplexes with high predicted efficacy,including maximal UA content in the seed region and low overall GCcontent.

For GalNaC-conjugated duplexes, sense 21mer and antisense 23mer oligoswere designed by extending antisense 19mers (described above) to 23nucleotides of target-complementary sequence. All species transcriptsincluded in the design batch were checked for complementarity. For eachduplex, the sense 21mer was specified as the reverse complement of thefirst 21 nucleotides of the antisense strand.

siRNA Sequence Selection

A total of 5 sense and 5 antisense human, 32 sense and 32 antisensederived human/cyno, 4 sense and 4 antisense derived human/cyno/mouse, 8sense and 8 antisense derived human/cyno/mouse/rat, 19 sense and 19antisense derived human/cyno/rat, 2 sense and 2 antisense derivedhuman/mouse, and 1 sense and 1 antisense derived human/mouse/rat siRNA21/23mer oligos were synthesized and formed into GalNAc-conjugatedduplexes.

The sequences of the sense and antisense strands of the unmodifiedduplexes are shown in Table 4, and the sequences of the sense andantisense strands of the modified duplexes are shown in Table 5.

TABLE 4 TMPRSS6-unmodified seqeunces Sense SEQ Antisense SEQ Positionsequence ID sequence ID in NM_ Duplex ID ID Sense sequence NO: IDAntisense sequence NO: 153609.2 AD-60944.1 A-122732.1GGUGCUACUCUGGUAUUUCCU 211 A-122733.1 AGGAAAUACCAGAGUAGCACCCC 280  318AD-59743.1 A-120243.1 UCUGGUAUUUCCUAGGGUACA 212 A-120244.1UGUACCCUAGGAAAUACCAGAGU 281  326 AD-60940.1 A-122745.1CUGGUAUUUCCUAGGGUACAA 213 A-122746.1 UUGUACCCUAGGAAAUACCAGAG 282  327AD-61002.2 A-122838.1 UGGUAUUUCCUAGGGUACAAA 214 A-122839.1UUUGUACCCUAGGAAAUACCAGA 283  328 AD-61000.1 A-122852.1GGUAUUUCCUAGGGUACAAGA 215 A-122853.1 UCUUGUACCCUAGGAAAUACCAG 284  329AD-46273.1 A-96908.1 UGGUAUUUCCUAGGGUACA 216 A-96909.1UGUACCCUAGGAAAUACCA 285  330 AD-61003.1 A-122854.1 GUAUUUCCUAGGGUACAAGGA217 A-122855.1 UCCUUGUACCCUAGGAAAUACCA 286  330 AD-60994.1 A-122848.1AUUUCCUAGGGUACAAGGCGA 218 A-122849.1 UCGCCUUGUACCCUAGGAAAUAC 287  332AD-60990.1 A-122830.1 UUUCCUAGGGUACAAGGCGGA 219 A-122831.1UCCGCCUUGUACCCUAGGAAAUA 288  333 AD-60956.1 A-122736.1CGCCACUUCUCCCAGGAUCUU 220 A-122737.1 AAGAUCCUGGGAGAAGUGGCGAU 289  400AD-60981.1 A-122757.1 GCCACUUCUCCCAGGAUCUUA 221 A-122758.1UAAGAUCCUGGGAGAAGUGGCGA 290  401 AD-60953.1 A-122775.1CUGCUUCUUCUGGUUCAUUCU 222 A-122776.1 AGAAUGAACCAGAAGAAGCAGGU 291  558AD-60977.1 A-122783.1 CUUCUUCUGGUUCAUUCUCCA 223 A-122784.1UGGAGAAUGAACCAGAAGAAGCA 292  561 AD-60964.1 A-119169.2CCCUACAGGGCCGAGUACGAA 224 A-122764.1 UUCGUACUCGGCCCUGUAGGGGA 293  679AD-60947.1 A-122773.1 CUACAGGGCCGAGUACGAAGU 225 A-122774.1ACUUCGUACUCGGCCCUGUAGGG 294  681 AD-60957.1 A-122751.1GCCAGUGUGAAAGACAUAGCU 226 A-122752.1 AGCUAUGUCUUUCACACUGGCUU 295  730AD-60960.1 A-122792.1 AGUGUGAAAGACAUAGCUGCA 227 A-122793.1UGCAGCUAUGUCUUUCACACUGG 296  733 AD-60972.1 A-122796.1CACGCUGGGUUGUUACCGCUA 228 A-122797.1 UAGCGGUAACAACCCAGCGUGGA 297  762AD-60970.1 A-122765.1 GGGUUGUUACCGCUACAGCUA 229 A-122766.1UAGCUGUAGCGGUAACAACCCAG 298  768 AD-60963.1 A-122753.1CGGGACCGACUGGCCAUGUAU 230 A-122754.1 AUACAUGGCCAGUCGGUCCCGGC 299  916AD-60968.1 A-122739.1 CCGACUGGCCAUGUAUGACGU 231 A-122740.1ACGUCAUACAUGGCCAGUCGGUC 300  921 AD-60942.1 A-122786.1GGGCCUGCACAGCUACUACGA 232 A-122787.1 UCGUAGUAGCUGUGCAGGCCCUU 301 1053AD-60951.1 A-122749.1 GGCAGAAGUAUGAUUUGCCGU 233 A-122750.1ACGGCAAAUCAUACUUCUGCCUC 302 1280 AD-60984.1 A-122800.1CCAGAACAGGAGGCUGUGUGG 234 A-122801.1 CCACACAGCCUCCUGUUCUGGAU 303 1323AD-60955.1 A-122806.1 CAGAACAGGAGGCUGUGUGGC 235 A-122807.1GCCACACAGCCUCCUGUUCUGGA 304 1324 AD-60943.1 A-122802.1CACCUCCCAGAUCUCCCUCAC 236 A-122803.1 GUGAGGGAGAUCUGGGAGGUGAA 305 1413AD-61001.1 A-122823.1 CACCUCCCAGAUCUCCCUCAA 237 A-122824.1UUGAGGGAGAUCUGGGAGGUGAA 306 1413 AD-60974.1 A-122741.1UGUGCGGGUGCACUAUGGCUU 238 A-122742.1 AAGCCAUAGUGCACCCGCACACC 307 1443AD-60982.1 A-122769.1 GCGGGUGCACUAUGGCUUGUA 239 A-122770.1UACAAGCCAUAGUGCACCCGCAC 308 1446 AD-60996.1 A-122834.1CCCCUGCCCUGGAGAGUUCCU 240 A-122835.1 AGGAACUCUCCAGGGCAGGGGUC 309 1479AD-60997.1 A-122850.1 CCCUGCCCUGGAGAGUUCCUA 241 A-122851.1UAGGAACUCUCCAGGGCAGGGGU 310 1480 AD-61006.1 A-122856.1CCUGCCCUGGAGAGUUCCUCU 242 A-122857.1 AGAGGAACUCUCCAGGGCAGGGG 311 1481AD-60988.1 A-122844.1 CUGCCCUGGAGAGUUCCUCUA 243 A-122845.1UAGAGGAACUCUCCAGGGCAGGG 312 1482 AD-60959.1 A-122777.1CCUGUGAUGGGGUCAAGGACU 244 A-122778.1 AGUCCUUGACCCCAUCACAGGCA 313 1529AD-60999.1 A-122836.1 GGACUGCCCCAACGGCCUGGA 245 A-122837.1UCCAGGCCGUUGGGGCAGUCCUU 314 1545 AD-60991.1 A-122846.1ACUGCCCCAACGGCCUGGAUA 246 A-122847.1 UAUCCAGGCCGUUGGGGCAGUCC 315 1547AD-60993.1 A-122832.1 CUGCCCCAACGGCCUGGAUGA 247 A-122833.1UCAUCCAGGCCGUUGGGGCAGUC 316 1548 AD-61005.1 A-122840.1UGCCCCAACGGCCUGGAUGAA 248 A-122841.1 UUCAUCCAGGCCGUUGGGGCAGU 317 1549AD-60987.1 A-119213.2 GCCCCAACGGCCUGGAUGAGA 249 A-122829.1UCUCAUCCAGGCCGUUGGGGCAG 318 1550 AD-60986.1 A-122842.1CCCCAACGGCCUGGAUGAGAA 250 A-122843.1 UUCUCAUCCAGGCCGUUGGGGCA 319 1551AD-60952.1 A-119187.2 CCCAACGGCCUGGAUGAGAGA 251 A-122761.1UCUCUCAUCCAGGCCGUUGGGGC 320 1552 AD-60983.1 A-119191.2CAACGGCCUGGAUGAGAGAAA 252 A-122785.1 UUUCUCUCAUCCAGGCCGUUGGG 321 1554AD-60950.1 A-122734.1 ACGGCCUGGAUGAGAGAAACU 253 A-122735.1AGUUUCUCUCAUCCAGGCCGUUG 322 1556 AD-60980.1 A-122743.1CCUGGAUGAGAGAAACUGCGU 254 A-122744.1 ACGCAGUUUCUCUCAUCCAGGCC 323 1560AD-60998.1 A-122821.1 CACUGUGACUGUGGCCUCCAA 255 A-122822.1UUGGAGGCCACAGUCACAGUGCU 324 1804 AD-60961.1 A-122808.1GUCCUCCGAGGGUGAGUGGCC 256 A-122809.1 GGCCACUCACCCUCGGAGGACAC 325 1857AD-61004.1 A-122825.1 CUCCGAGGGUGAGUGGCCAUA 257 A-122826.1UAUGGCCACUCACCCUCGGAGGA 326 1860 AD-60949.1 A-122804.1UCCGAGGGUGAGUGGCCAUGG 258 A-122805.1 CCAUGGCCACUCACCCUCGGAGG 327 1861AD-60969.1 A-119189.2 CCAGGUUCGGGGUCGACACAU 259 A-122755.1AUGUGUCGACCCCGAACCUGGAG 328 1893 AD-60966.1 A-122794.1AGGUUCGGGGUCGACACAUCU 260 A-122795.1 AGAUGUGUCGACCCCGAACCUGG 329 1895AD-60967.1 A-122810.1 CGGGGUCGACACAUCUGUGGG 261 A-122811.1CCCACAGAUGUGUCGACCCCGAA 330 1900 AD-60989.1 A-122816.1CGGGGUCGACACAUCUGUGGA 262 A-122817.1 UCCACAGAUGUGUCGACCCCGAA 331 1900AD-60973.1 A-122812.1 GGGGUCGACACAUCUGUGGGG 263 A-122813.1CCCCACAGAUGUGUCGACCCCGA 332 1901 AD-60992.1 A-122818.1GGGGUCGACACAUCUGUGGGA 264 A-122819.1 UCCCACAGAUGUGUCGACCCCGA 333 1901AD-60985.1 A-122827.1 GGGUCGACACAUCUGUGGGGA 265 A-122828.1UCCCCACAGAUGUGUCGACCCCG 334 1902 AD-60946.1 A-122759.1GCUGACCGCUGGGUGAUAACA 266 A-122760.1 UGUUAUCACCCAGCGGUCAGCGA 335 1933AD-60979.1 A-122814.1 CUUCCAGGAGGACAGCAUGGC 267 A-122815.1GCCAUGCUGUCCUCCUGGAAGCA 336 1965 AD-60976.1 A-122767.1GGCCUGGAGAGGUGUCCUUCA 268 A-122768.1 UGAAGGACACCUCUCCAGGCCAG 337 2039AD-60939.1 A-122730.1 GCCUGGAGAGGUGUCCUUCAA 269 A-122731.1UUGAAGGACACCUCUCCAGGCCA 338 2040 AD-60978.1 A-122798.1CCAAGCAGGGGGACAAGUAUU 270 A-122799.1 AAUACUUGUCCCCCUGCUUGGCA 339 2608AD-60958.1 A-122762.1 CAAGCAGGGGGACAAGUAUUC 271 A-122763.1GAAUACUUGUCCCCCUGCUUGGC 340 2609 AD-60962.1 A-119231.2UGGCAGGAGGUGGCAUCUUGU 272 A-122738.1 ACAAGAUGCCACCUCCUGCCACC 341 2664AD-60941.1 A-122771.1 GCAGGAGGUGGCAUCUUGUCU 273 A-122772.1AGACAAGAUGCCACCUCCUGCCA 342 2666 AD-60965.1 A-122779.1GCUUCGGAAGCCCCUGGUCUA 274 A-122780.1 UAGACCAGGGGCUUCCGAAGCUG 343 2954AD-60954.1 A-122790.1 CUUCGGAAGCCCCUGGUCUAA 275 A-122791.1UUAGACCAGGGGCUUCCGAAGCU 344 2955 AD-60975.1 A-119233.2CCCCUGGUCUAACUUGGGAUC 276 A-122756.1 GAUCCCAAGUUAGACCAGGGGCU 345 2964AD-60945.1 A-122747.1 CCCUGGUCUAACUUGGGAUCU 277 A-122748.1AGAUCCCAAGUUAGACCAGGGGC 346 2965 AD-60971.1 A-122781.1CCUGGUCUAACUUGGGAUCUG 278 A-122782.1 CAGAUCCCAAGUUAGACCAGGGG 347 2966AD-60948.1 A-122788.1 CUAACUUGGGAUCUGGGAAUG 279 A-122789.1CAUUCCCAGAUCCCAAGUUAGAC 348 2972

TABLE 5 TMPRSS6 modified sequences SEQ SEQ Duplex Sense  ID Antisense IDID sequence ID Sense sequence NO: sequence ID Antisense sequence NO:AD-46273.1 A-96908.1 uGGuAuuuccuAGGGuAcAdTsdT 349 A-96909.1UGuACCCuAGGAAAuACcAdTsdT 418 AD-59743.1 A-120243.1UfscsUfgGfuAfuUfUfCfcUfaGfgGfuAfcAfL96 350 A-120244.1usGfsuAfcCfcUfaGfgaaAfuAfcCfaGfasgsu 419 AD-60939.1 A-122730.1GfscsCfuGfgAfgAfGfGfuGfuCfcUfuCfaAfL96 351 A-122731.1usUfsgAfaGfgAfcAfccuCfuCfcAfgGfcscsa 420 AD-60940.1 A-122745.1CfsusGfgUfaUfuUfCfCfuAfgGfgUfaCfaAfL96 352 A-122746.1usUfsgUfaCfcCfuAfggaAfaUfaCfcAfgsasg 421 AD-60941.1 A-122771.1GfscsAfgGfaGfgUfGfGfcAfuCfuUfgUfcUfL96 353 A-122772.1asGfsaCfaAfgAfuGfccaCfcUfcCfuGfcscsa 422 AD-60942.1 A-122786.1GfsgsGfcCfuGfcAfCfAfgCfuAfcUfaCfgAfL96 354 A-122787.1usCfsgUfaGfuAfgCfuguGfcAfgGfcCfcsusu 423 AD-60943.1 A-122802.1CfsasCfcUfcCfcAfGfAfuCfuCfcCfuCfaCfL96 355 A-122803.1gsUfsgAfgGfgAfgAfucuGfgGfaGfgUfgsasa 424 AD-60944.1 A-122732.1GfsgsUfgCfuAfcUfCfUfgGfuAfuUfuCfcUfL96 356 A-122733.1asGfsgAfaAfuAfcCfagaGfuAfgCfaCfcscsc 425 AD-60945.1 A-122747.1CfscsCfuGfgUfcUfAfAfcUfuGfgGfaUfcUfL96 357 A-122748.1asGfsaUfcCfcAfaGfuuaGfaCfcAfgGfgsgsc 426 AD-60946.1 A-122759.1GfscsUfgAfcCfgCfUfGfgGfuGfaUfaAfcAfL96 358 A-122760.1usGfsuUfaUfcAfcCfcagCfgGfuCfaGfcsgsa 427 AD-60947.1 A-122773.1CfsusAfcAfgGfgCfCfGfaGfuAfcGfaAfgUfL96 359 A-122774.1asCfsuUfcGfuAfcUfcggCfcCfuGfuAfgsgsg 428 AD-60948.1 A-122788.1CfsusAfaCfuUfgGfGfAfuCfuGfgGfaAfuGfL96 360 A-122789.1csAfsuUfcCfcAfgAfuccCfaAfgUfuAfgsasc 429 AD-60949.1 A-122804.1UfscsCfgAfgGfgUfGfAfgUfgGfcCfaUfgGfL96 361 A-122805.1csCfsaUfgGfcCfaCfucaCfcCfuCfgGfasgsg 430 AD-60950.1 A-122734.1AfscsGfgCfcUfgGfAfUfgAfgAfgAfaAfcUfL96 362 A-122735.1asGfsuUfuCfuCfuCfaucCfaGfgCfcGfususg 431 AD-60951.1 A-122749.1GfsgsCfaGfaAfgUfAfUfgAfuUfuGfcCfgUfL96 363 A-122750.1asCfsgGfcAfaAfuCfauaCfuUfcUfgCfcsusc 432 AD-60952.1 A-119187.2CfscsCfaAfcGfgCfCfUfgGfaUfgAfgAfgAfL96 364 A-122761.1usCfsuCfuCfaUfcCfaggCfcGfuUfgGfgsgsc 433 AD-60953.1 A-122775.1CfsusGfcUfuCfuUfCfUfgGfuUfcAfuUfcUfL96 365 A-122776.1asGfsaAfuGfaAfcCfagaAfgAfaGfcAfgsgsu 434 AD-60954.1 A-122790.1CfsusUfcGfgAfaGfCfCfcCfuGfgUfcUfaAfL96 366 A-122791.1usUfsaGfaCfcAfgGfggcUfuCfcGfaAfgscsu 435 AD-60955.1 A-122806.1CfsasGfaAfcAfgGfAfGfgCfuGfuGfuGfgCfL96 367 A-122807.1gsCfscAfcAfcAfgCfcucCfuGfuUfcUfgsgsa 436 AD-60956.1 A-122736.1CfsgsCfcAfcUfuCfUfCfcCfaGfgAfuCfuUfL96 368 A-122737.1asAfsgAfuCfcUfgGfgagAfaGfuGfgCfgsasu 437 AD-60957.1 A-122751.1GfscsCfaGfuGfuGfAfAfaGfaCfaUfaGfcUfL96 369 A-122752.1asGfscUfaUfgUfcUfuucAfcAfcUfgGfcsusu 438 AD-60958.1 A-122762.1CfsasAfgCfaGfgGfGfGfaCfaAfgUfaUfuCfL96 370 A-122763.1gsAfsaUfaCfuUfgUfcccCfcUfgCfuUfgsgsc 439 AD-60959.1 A-122777.1CfscsUfgUfgAfuGfGfGfgUfcAfaGfgAfcUfL96 371 A-122778.1asGfsuCfcUfuGfaCfcccAfuCfaCfaGfgscsa 440 AD-60960.1 A-122792.1AfsgsUfgUfgAfaAfGfAfcAfuAfgCfuGfcAfL96 372 A-122793.1usGfscAfgCfuAfuGfucuUfuCfaCfaCfusgsg 441 AD-60961.1 A-122808.1GfsusCfcUfcCfgAfGfGfgUfgAfgUfgGfcCfL96 373 A-122809.1gsGfscCfaCfuCfaCfccuCfgGfaGfgAfcsasc 442 AD-60962.1 A-119231.2UfsgsGfcAfgGfaGfGfUfgGfcAfuCfuUfgUfL96 374 A-122738.1asCfsaAfgAfuGfcCfaccUfcCfuGfcCfascsc 443 AD-60963.1 A-122753.1CfsgsGfgAfcCfgAfCfUfgGfcCfaUfgUfaUfL96 375 A-122754.1asUfsaCfaUfgGfcCfaguCfgGfuCfcCfgsgsc 444 AD-60964.1 A-119169.2CfscsCfuAfcAfgGfGfCfcGfaGfuAfcGfaAfL96 376 A-122764.1usUfscGfuAfcUfcGfgccCfuGfuAfgGfgsgsa 445 AD-60965.1 A-122779.1GfscsUfuCfgGfaAfGfCfcCfcUfgGfuCfuAfL96 377 A-122780.1usAfsgAfcCfaGfgGfgcuUfcCfgAfaGfcsusg 446 AD-60966.1 A-122794.1AfsgsGfuUfcGfgGfGfUfcGfaCfaCfaUfcUfL96 378 A-122795.1asGfsaUfgUfgUfcGfaccCfcGfaAfcCfusgsg 447 AD-60967.1 A-122810.1CfsgsGfgGfuCfgAfCfAfcAfuCfuGfuGfgGfL96 379 A-122811.1csCfscAfcAfgAfuGfuguCfgAfcCfcCfgsasa 448 AD-60968.1 A-122739.1CfscsGfaCfuGfgCfCfAfuGfuAfuGfaCfgUfL96 380 A-122740.1asCfsgUfcAfuAfcAfuggCfcAfgUfcGfgsusc 449 AD-60969.1 A-119189.2CfscsAfgGfuUfcGfGfGfgUfcGfaCfaCfaUfL96 381 A-122755.1asUfsgUfgUfcGfaCfcccGfaAfcCfuGfgsasg 450 AD-60970.1 A-122765.1GfsgsGfuUfgUfuAfCfCfgCfuAfcAfgCfuAfL96 382 A-122766.1usAfsgCfuGfuAfgCfgguAfaCfaAfcCfcsasg 451 AD-60971.1 A-122781.1CfscsUfgGfuCfuAfAfCfuUfgGfgAfuCfuGfL96 383 A-122782.1csAfsgAfuCfcCfaAfguuAfgAfcCfaGfgsgsg 452 AD-60972.1 A-122796.1CfsasCfgCfuGfgGfUfUfgUfuAfcCfgCfuAfL96 384 A-122797.1usAfsgCfgGfuAfaCfaacCfcAfgCfgUfgsgsa 453 AD-60973.1 A-122812.1GfsgsGfgUfcGfaCfAfCfaUfcUfgUfgGfgGfL96 385 A-122813.1csCfscCfaCfaGfaUfgugUfcGfaCfcCfcsgsa 454 AD-60974.1 A-122741.1UfsgsUfgCfgGfgUfGfCfaCfuAfuGfgCfuUfL96 386 A-122742.1asAfsgCfcAfuAfgUfgcaCfcCfgCfaCfascsc 455 AD-60975.1 A-119233.2CfscsCfcUfgGfuCfUfAfaCfuUfgGfgAfuCfL96 387 A-122756.1gsAfsuCfcCfaAfgUfuagAfcCfaGfgGfgscsu 456 AD-60976.1 A-122767.1GfsgsCfcUfgGfaGfAfGfgUfgUfcCfuUfcAfL96 388 A-122768.1usGfsaAfgGfaCfaCfcucUfcCfaGfgCfcsasg 457 AD-60977.1 A-122783.1CfsusUfcUfuCfuGfGfUfuCfaUfuCfuCfcAfL96 389 A-122784.1usGfsgAfgAfaUfgAfaccAfgAfaGfaAfgscsa 458 AD-60978.1 A-122798.1CfscsAfaGfcAfgGfGfGfgAfcAfaGfuAfuUfL96 390 A-122799.1asAfsuAfcUfuGfuCfcccCfuGfcUfuGfgscsa 459 AD-60979.1 A-122814.1CfsusUfcCfaGfgAfGfGfaCfaGfcAfuGfgCfL96 391 A-122815.1gsCfscAfuGfcUfgUfccuCfcUfgGfaAfgscsa 460 AD-60980.1 A-122743.1CfscsUfgGfaUfgAfGfAfgAfaAfcUfgCfgUfL96 392 A-122744.1asCfsgCfaGfuUfuCfucuCfaUfcCfaGfgscsc 461 AD-60981.1 A-122757.1GfscsCfaCfuUfcUfCfCfcAfgGfaUfcUfuAfL96 393 A-122758.1usAfsaGfaUfcCfuGfggaGfaAfgUfgGfcsgsa 462 AD-60982.1 A-122769.1GfscsGfgGfuGfcAfCfUfaUfgGfcUfuGfuAfL96 394 A-122770.1usAfscAfaGfcCfaUfaguGfcAfcCfcGfcsasc 463 AD-60983.1 A-119191.2CfsasAfcGfgCfcUfGfGfaUfgAfgAfgAfaAfL96 395 A-122785.1usUfsuCfuCfuCfaUfccaGfgCfcGfuUfgsgsg 464 AD-60984.1 A-122800.1CfscsAfgAfaCfaGfGfAfgGfcUfgUfgUfgGfL96 396 A-122801.1csCfsaCfaCfaGfcCfuccUfgUfuCfuGfgsasu 465 AD-60985.1 A-122827.1GfsgsGfuCfgAfcAfCfAfuCfuGfuGfgGfgAfL96 397 A-122828.1usCfscCfcAfcAfgAfuguGfuCfgAfcCfcscsg 466 AD-60986.1 A-122842.1CfscsCfcAfaCfgGfCfCfuGfgAfuGfaGfaAfL96 398 A-122843.1usUfscUfcAfuCfcAfggcCfgUfuGfgGfgscsa 467 AD-60987.1 A-119213.2GfscsCfcCfaAfcGfGfCfcUfgGfaUfgAfgAfL96 399 A-122829.1usCfsuCfaUfcCfaGfgccGfuUfgGfgGfcsasg 468 AD-60988.1 A-122844.1CfsusGfcCfcUfgGfAfGfaGfuUfcCfuCfuAfL96 400 A-122845.1usAfsgAfgGfaAfcUfcucCfaGfgGfcAfgsgsg 469 AD-60989.1 A-122816.1CfsgsGfgGfuCfgAfCfAfcAfuCfuGfuGfgAfL96 401 A-122817.1usCfscAfcAfgAfuGfuguCfgAfcCfcCfgsasa 470 AD-60990.1 A-122830.1UfsusUfcCfuAfgGfGfUfaCfaAfgGfcGfgAfL96 402 A-122831.1usCfscGfcCfuUfgUfaccCfuAfgGfaAfasusa 471 AD-60991.1 A-122846.1AfscsUfgCfcCfcAfAfCfgGfcCfuGfgAfuAfL96 403 A-122847.1usAfsuCfcAfgGfcCfguuGfgGfgCfaGfuscsc 472 AD-60992.1 A-122818.1GfsgsGfgUfcGfaCfAfCfaUfcUfgUfgGfgAfL96 404 A-122819.1usCfscCfaCfaGfaUfgugUfcGfaCfcCfcsgsa 473 AD-60993.1 A-122832.1CfsusGfcCfcCfaAfCfGfgCfcUfgGfaUfgAfL96 405 A-122833.1usCfsaUfcCfaGfgCfcguUfgGfgGfcAfgsusc 474 AD-60994.1 A-122848.1AfsusUfuCfcUfaGfGfGfuAfcAfaGfgCfgAfL96 406 A-122849.1usCfsgCfcUfuGfuAfcccUfaGfgAfaAfusasc 475 AD-60996.1 A-122834.1CfscsCfcUfgCfcCfUfGfgAfgAfgUfuCfcUfL96 407 A-122835.1asGfsgAfaCfuCfuCfcagGfgCfaGfgGfgsusc 476 AD-60997.1 A-122850.1CfscsCfuGfcCfcUfGfGfaGfaGfuUfcCfuAfL96 408 A-122851.1usAfsgGfaAfcUfcUfccaGfgGfcAfgGfgsgsu 477 AD-60998.1 A-122821.1CfsasCfuGfuGfaCfUfGfuGfgCfcUfcCfaAfL96 409 A-122822.1usUfsgGfaGfgCfcAfcagUfcAfcAfgUfgscsu 478 AD-60999.1 A-122836.1GfsgsAfcUfgCfcCfCfAfaCfgGfcCfuGfgAfL96 410 A-122837.1usCfscAfgGfcCfgUfuggGfgCfaGfuCfcsusu 479 AD-61000.1 A-122852.1GfsgsUfaUfuUfcCfUfAfgGfgUfaCfaAfgAfL96 411 A-122853.1usCfsuUfgUfaCfcCfuagGfaAfaUfaCfcsasg 480 AD-61001.1 A-122823.1CfsasCfcUfcCfcAfGfAfuCfuCfcCfuCfaAfL96 412 A-122824.1usUfsgAfgGfgAfgAfucuGfgGfaGfgUfgsasa 481 AD-61002.1 A-122838.1UfsgsGfuAfuUfuCfCfUfaGfgGfuAfcAfaAfL96 413 A-122839.1usUfsuGfuAfcCfcUfaggAfaAfuAfcCfasgsa 482 AD-61003.1 A-122854.1GfsusAfuUfuCfcUfAfGfgGfuAfcAfaGfgAfL96 414 A-122855.1usCfscUfuGfuAfcCfcuaGfgAfaAfuAfcscsa 483 AD-61004.1 A-122825.1CfsusCfcGfaGfgGfUfGfaGfuGfgCfcAfuAfL96 415 A-122826.1usAfsuGfgCfcAfcUfcacCfcUfcGfgAfgsgsa 484 AD-61005.1 A-122840.1UfsgsCfcCfcAfaCfGfGfcCfuGfgAfuGfaAfL96 416 A-122841.1usUfscAfuCfcAfgGfccgUfuGfgGfgCfasgsu 485 AD-61006.1 A-122856.1CfscsUfgCfcCfuGfGfAfgAfgUfuCfcUfcUfL96 417 A-122857.1asGfsaGfgAfaCfuCfuccAfgGfgCfaGfgsgsg 486

Example 6. In Vitro Single Dose Screen Cell Culture and Transfectionsfor Single Dose and Dose Response Studies

Hep3B cells (ATCC, Manassas, Va.) were grown to near confluence at 37°C. in an atmosphere of 5% CO₂ in DMEM (ATCC) supplemented with 10% FBS,streptomycin, and glutamine (ATCC) before being released from the plateby trypsinization. Transfection was carried out by adding 14.8 μl ofOpti-MEM plus 0.2 μl of Lipofectamine RNAiMax per well (Invitrogen,Carlsbad Calif. cat #13778-150) to 5 μl of siRNA duplexes per well intoa 96-well plate and incubated at room temperature for 15 minutes. 80 μlof complete growth media without antibiotic containing ˜2×10⁴ Hep3Bcells were then added to the siRNA mixture. Cells were incubated for 24hours prior to RNA purification. Experiments were performed at 10 nM and0.1 nM final duplex concentration.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit (Invitrogen, Part#: 610-12)

Cells were harvested and lysed in 150 μl of Lysis/Binding Buffer thenmixed for 5 minutes at 850 rpm using an Eppendorf Thermomixer (themixing speed was the same throughout the process). Ten microliters ofmagnetic beads and 80 μl Lysis/Binding Buffer mixture were added to around bottom plate and mixed for 1 minute. Magnetic beads were capturedusing magnetic stand and the supernatant was removed without disturbingthe beads. After removing supernatant, the lysed cells were added to theremaining beads and mixed for 5 minutes. After removing supernatant,magnetic beads were washed 2 times with 150 μl Wash Buffer A and mixedfor 1 minute. Beads were capture again and supernatant removed. Beadswere then washed with 150 μl Wash Buffer B, captured and supernatant wasremoved. Beads were next washed with 150 μl Elution Buffer, captured andsupernatant removed. Beads were allowed to dry for 2 minutes. Afterdrying, 50 μl of Elution Buffer was added and mixed for 5 minutes at 70°C. Beads were captured on magnet for 5 minutes. 40 μl of supernatant wasremoved and added to another 96 well plate.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit(Applied Biosystems, Foster City, Calif., Cat #4368813)

A master mix of 2 μl 10× Buffer, 0.8 μl 25×dNTPs, 2 μl Random primers, 1μl Reverse Transcriptase, 1 μl RNase inhibitor and 3.2 μl of H2O perreaction were added into 101 total RNA. cDNA was generated using aBio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through thefollowing steps: 25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C.hold.

Real Time PCR

2 μl of cDNA were added to a master mix containing 0.5 μl GAPDH TaqManProbe (Applied Biosystems Cat #4326317E), 0.5 μl TMPRSS6 TaqMan probe(Applied Biosystems cat # Hs00542184_m1) and 5 μl Lightcycler 480 probemaster mix (Roche Cat #04887301001) per well in a 384 well 50 plates(Roche cat #04887301001). Real time PCR was done in an Roche LightcyclerReal Time PCR system (Roche) using the ΔΔCt(RQ) assay. Each duplex wastested in two independent transfections and each transfection wasassayed in duplicate, unless otherwise noted in the summary tables.

To calculate relative fold change, real time data were analyzed usingthe ΔΔCt method and normalized to assays performed with cellstransfected with 10 nM AD-1955, or mock transfected cells.

Data are expressed as a fraction of TMPRSS6 message remaining in cellstransfected with siRNAs targeting TMPRSS6, relative to naïve cells. AllsiRNAs were transfected at least two times and qPCR reactions wereperformed in duplicate. Data are show in Table 6.

TABLE 6 TMPRSS6 single dose screen. Duplex ID Avg 10 nM Avg 0.1 nM SD 10nM SD 0.1 nM AD-46273 76.5 112.1 14.3 18.6 AD-59743 61.4 108.2 8.7 4.4AD-60939 38.0 85.7 19.3 25.2 AD-60940 24.2 22.6 10.1 9.7 AD-60941 48.584.7 11.7 29.7 AD-60942 102.9 111.2 4.3 44.8 AD-60943 86.2 96.5 2.3 28.8AD-60944 24.6 78.5 1.1 36.5 AD-60945 65.8 140.9 0.5 59.2 AD-60946 50.3105.9 4.1 31.2 AD-60947 79.1 147.2 12.3 51.2 AD-60948 81.0 113.9 0.632.7 AD-60949 111.3 96.2 8.2 28.1 AD-60950 53.8 93.2 7.6 42.3 AD-6095174.1 121.6 6.4 56.2 AD-60952 47.6 118.3 8.1 52.4 AD-60953 22.0 56.7 8.318.0 AD-60954 23.3 55.8 5.3 31.7 AD-60955 110.8 117.5 1.6 38.7 AD-6095615.8 29.6 1.7 10.2 AD-60957 22.3 58.3 1.5 6.1 AD-60958 106.4 136.0 24.161.7 AD-60959 79.6 123.3 0.6 49.9 AD-60960 17.4 49.4 8.6 10.2 AD-60961107.7 129.0 6.6 50.5 AD-60962 90.2 113.3 8.0 67.2 AD-60963 117.4 138.12.6 16.8 AD-60964 80.7 123.2 24.2 18.9 AD-60965 30.1 80.2 9.0 20.8AD-60966 54.1 133.6 4.6 44.0 AD-60967 122.2 147.4 11.7 42.0 AD-6096886.9 142.0 39.9 49.7 AD-60969 106.2 116.3 16.6 39.1 AD-60970 54.6 112.67.3 11.8 AD-60971 50.5 118.8 6.9 47.0 AD-60972 55.6 94.2 6.5 3.4AD-60973 126.1 133.6 8.0 36.8 AD-60974 82.6 115.0 8.7 43.7 AD-60975 88.2114.3 13.6 43.9 AD-60976 46.3 71.0 11.6 30.2 AD-60977 13.5 26.4 3.4 9.2AD-60978 72.7 92.9 6.4 31.7 AD-60979 103.8 97.0 13.7 29.2 AD-60980 28.458.0 12.3 21.1 AD-60981 56.0 80.6 18.3 4.5 AD-60982 102.4 137.4 15.216.4 AD-60983 60.8 87.1 10.1 20.3 AD-60984 53.6 116.7 1.2 47.8 AD-6098572.6 99.2 0.7 21.7 AD-60986 90.1 96.4 6.6 29.5 AD-60987 83.1 90.7 1.613.7 AD-60988 69.4 102.3 2.4 55.4 AD-60989 112.4 105.7 0.6 14.7 AD-6099090.4 93.4 6.2 4.1 AD-60991 97.6 95.6 15.5 23.4 AD-60992 104.0 131.4 6.933.7 AD-60993 118.6 129.2 10.5 30.1 AD-60994 25.9 57.2 6.8 0.3 AD-6099677.3 94.2 7.8 12.6 AD-60997 60.1 80.9 18.8 7.5 AD-60998 32.6 61.4 5.724.6 AD-60999 133.6 110.9 39.7 15.4 AD-61000 55.8 117.6 14.2 24.9AD-61001 57.9 85.2 8.1 42.0 AD-61002 15.4 31.4 1.5 10.1 AD-61003 82.398.1 4.0 11.8 AD-61004 106.4 97.7 38.5 18.8 AD-61005 138.0 141.2 65.720.0 AD-61006 31.7 70.9 7.8 6.6

Example 7. In Vivo Effect of Single Dose Administration of TMPRSS6 iRNAAgent

Female C57BL/6 mice were administered a single subcutaneous injection ofAD-60940 at a dose of 0.3 mg/kg, 1.0 mg/kg or 3.0 mg/kg, or PBS alone asa control. Three mice were evaluated per dose for hepatic TMPRSS6 mRNA,hepatic hepcidin mRNA, serum hepcidin, total serum iron, and percenttransferrin saturation at various time points. Mice receiving 1.0 mg/kgor 3.0 mg/kg of AD-60940 or PBS were evaluated at day 0 (pre-treatment)and 7, 11, 14 and 21 days after treatment. Mice receiving 0.3 mg/kgAD-60940 were evaluated at day 0 (pre-treatment) and at 7 and 11 daysafter treatment. Hepatic TMPRSS6 mRNA and hepatic hepcidin mRNA levelswere determined by qPCR, normalized to GAPDH mRNA levels and expressedrelative to the mRNA levels in mice administered PBS alone. Serumhepcidin was measured by ELISA (Intrinsic Life Sciences). Total serumiron and percent transferrin saturation (% TfSat) were measured using anOlympus AU400 Serum Chemistry Analyzer. Each data point represents themean value from three mice. The standard deviation of the mean isrepresented by error bars.

Single dose administration of AD-60940 resulted in robust and durablesuppression of hepatic TMPRSS6 mRNA relative to the control. TMPRSS6mRNA concentration was suppressed by greater than 90% for up to threeweeks following administration of the 3.0 mg/kg dose (FIG. 3A). As aresult of the suppression of hepatic TMPRSS6 mRNA concentration,hepcidin mRNA levels, increased two-fold relative to the control (FIG.3B), and serum hepcidin concentration increased greater than 2-foldrelative to the control (FIG. 3C). In addition, total serum iron (FIG.3D) decreased and percent transferrin saturation decreased by greaterthan 50% relative to the control (FIG. 3E). The decreases in total serumiron and percent transferring saturation were durable for up to threeweeks following administration of AD-60940. FIG. 3F demonstrates therelative hepatic TMPRSS6 mRNA concentration as a function of AD-60940dose at 11 days following administration. Each data point represents themaximum suppression of TMPRSS6 mRNA concentration observed at each doselevel. The data were fit to the Hill equation.

The degree to which AD-60940 modulates hepcidin and serum ironmobilization is nearly identical to that observed in the previousHbb^(th3/+) mouse studies (Schmidt et al., Blood (2013), 121(7),1200-1208) and indicates that AD-60940 is a potent RNAi therapeutic forproducing disease modifying effects in β-Thalassemia.

Example 8. In Vivo Effect of Multi-Dose Administration of TMPRSS6 iRNAAgent

Female C57BL/6 mice were administered a subcutaneous injection ofAD-60940 at a dose of 0.3 mg/kg, 1.0 mg/kg, or PBS alone (as a control)once per week for three weeks then sacrificed 7 days after the finaldose (FIG. 4A). Three mice per dose were evaluated for hepatic TMPRSS6mRNA, hepatic hepcidin mRNA, and percent transferrin saturation. HepaticTMPRSS6 mRNA and hepatic hepcidin mRNA levels were determined by qPCR,normalized to GAPDH mRNA levels and expressed relative to the mRNAlevels in mice administered PBS alone. Percent transferrin saturation (%TfSat) was measured using an Olympus AU400 Serum Chemistry Analyzer.Each data point represents the mean value from three mice. The standarddeviation of the mean is represented by error bars.

Multi-dose administration of 1.0 mg/kg AD-60940 resulted in greater than90% suppression of TMPRSS6 mRNA concentration (FIG. 4B). Hepcidin mRNAconcentration increased two-fold and percent transferrin saturationdecreased by greater than 50% relative to the control (FIG. 4B). FIG. 4Cdemonstrates the relative hepatic TMPRSS6 mRNA concentration as afunction of AD-60940 dose. The data were fit to the Hill equation. Thesedata indicate that the multi-dose ED80 is less than 1.0 mg/kg.

This study demonstrates that AD-60940 exhibits robust and durablesuppression of TMPRSS6, resulting in hepcidin induction and systemiciron restriction and indicates that AD-60940 is a potent RNAitherapeutic for producing disease modifying effects in β-Thalassemia.

Example 9. Relationship Between Liver TMPRSS6 mRNA Levels and SerumHepcidin Concentration and Percent Transferrin Saturation

Data generated using AD-59743, AD-61002, AD-60940, and other TMPRSS6iRNA agents were further analyzed to evaluate the relationship betweenliver TMPRSS6 mRNA levels and serum hepcidin levels and percenttransferrin saturation. Serum hepcidin concentration demonstrated anon-linear relationship to TMPRSS6 mRNA levels using the Hill equation(FIG. 5A). The percent transferrin saturation demonstrated a linearrelationship to TMPRSS6 mRNA levels when fit to a simple linearregression equation (FIG. 5B). The linear relationship between TMPRSS6mRNA levels and percent transferrin saturation indicate that ironrestriction can be precisely and predictably modulated by AD-60940.Serum hepcidin concentration and relative hepcidin mRNA levels alsodemonstrated a linear relationship when fit to a simple linearregression equation (FIG. 5C). In contrast, the relationship betweenpercent transferrin saturation and serum hepcidin concentration wasnon-linear and fit to the Hill equation (FIG. 5D).

Example 10. In Vivo Single Dose Screen

TMPRSS6 siRNA duplexes as indicated in FIG. 6 were evaluated forefficacy by their ability to suppress levels of TMPRSS6 mRNA in theliver of female C57BL/6 mice following administration of the siRNAduplex. A single subcutaneous dose of 3 mg/kg of TMPRSS6 siRNA duplexwas administered, and the mice were sacrificed 7 days later. The levelof TMPRSS6 mRNA in the liver was measured by qPCR using the methodsdescribed above. Mice in a control group received an injection of PBS.

The levels of TMPRSS6 mRNA following administration of a TMPRSS6 siRNAduplex are shown in FIG. 6. The results demonstrate that administrationof AD-60940, AD-59743 and AD-61002 resulted in substantial suppressionof liver TMPRSS6 mRNA with AD60940 producing the greatest silencing.Specifically, TMPRSS6 siRNA duplex AD-60940 reduced TMPRSS6 mRNA bygreater than 80% relative to the control. The data also demonstrate thattreatment with AD-59743, AD-60940, AD-61002, AD-60994, AD-60998 andAD-61001 result in a decrease in the level of TMPRSS6 transcript that ismaintained through day 7.

Example 11. In Vivo Multi-Dose Screen

TMPRSS6 siRNA duplexes as indicated in FIG. 7 were evaluated forefficacy by their ability to suppress levels of TMPRSS6 mRNA in theliver of wild-type C57BL/6 mice following administration of the siRNAduplex. A subcutaneous dose of either 0.3 mg/kg or 1.0 mg/kg of TMPRSS6siRNA duplex was administered once a week for three weeks. The mice weresacrificed 7 days after the last dose. The level of TMPRSS6 mRNA in theliver was measured by qPCR using the methods described above. Mice in acontrol group received an injection of PBS.

The levels of TMPRSS6 mRNA following administration of a TMPRSS6 siRNAduplex are shown in FIG. 7. The results demonstrate that the 1.0 mg/kgdosing regimen of TMPRSS6 siRNA duplex AD-60940 reduces TMPRSS6 mRNA bygreater than 80% relative to the control.

Example 12. Optimization of AD-60940

Based on the observation that administration of AD-60940 durably reducedTMPRSS6 mRNA by greater than 80% relative to the control, additionalsiRNAs based on the parent sequence of AD-60940 with a variety ofchemical modifications were evaluated for efficacy in single dosescreens at 10 nM and 0.1 nM by transfection in Hep3B cells. Thesequences of the sense and antisense strands of these agents are shownin Table 8 and the results of this screen are shown in Table 9. The datain Table 9 are expressed as the average fraction message remainingrelative to control.

In addition, a subset of siRNA described in Tables 4 and 5, above, weremodified to replace a 2′F with a 2′OMe modification at the 5′-end of thesense strand and to add a 5′-phosphate on the antisense strand. ThesesiRNA agents were also evaluated for in vitro efficacy in single dosescreens at 10 nM and 0.1 nM by transfection in Hep3B cells. Thesequences of the sense and antisense strands of these agents are shownin Table 10 and the results of this screen are shown in Table 11. Thedata in Table 11 are expressed as the average fraction message remainingrelative to control.

TABLE 8 TMPRSS6 Modified Sequences SEQ SEQ ID ID DuplexID SenseIDSense Sequence NO: AntisenseID Antisense Sequence NO: AD-63214A-126586.2 Y44CfsusGfgUfaUfuUfCfCfuAfgGfgUfaCfaAfL96 487 A-126587.2PusUfsgUfaCfcCfuAfggaAfaUfaCfcAfgsasg 544 AD-63240 A-122745.11CfsusGfgUfaUfuUfCfCfuAfgGfgUfaCfaAfL96 488 A-126607.1usUfsguaCfcCfuAfggaAfaUfaccagsasg 545 AD-63209 A-126594.1csusgguaUfuUfCfCfuaggGfdTacaaL96 489 A-122746.13usUfsgUfaCfcCfuAfggaAfaUfaCfcAfgsasg 546 AD-63208 A-122745.6CfsusGfgUfaUfuUfCfCfuAfgGfgUfaCfaAfL96 490 A-126587.1PusUfsgUfaCfcCfuAfggaAfaUfaCfcAfgsasg 547 AD-63202 A-126586.1Y44CfsusGfgUfaUfuUfCfCfuAfgGfgUfaCfaAfL96 491 A-122746.6usUfsgUfaCfcCfuAfggaAfaUfaCfcAfgsasg 548 AD-63216 A-122745.7CfsusGfgUfaUfuUfCfCfuAfgGfgUfaCfaAfL96 492 A-126603.1usUfsgUfaCfccuAfggaAfaUfaCfcAfgsasg 549 AD-63219 A-126617.1gsgsUfaUfuUfCfCfuAfgGfgUfaCfaAfL96 493 A-126618.1PusUfsgUfaCfcCfuAfggaAfaUfaCfcsasg 550 AD-63228 A-122745.9CfsusGfgUfaUfuUfCfCfuAfgGfgUfaCfaAfL96 494 A-126605.1usUfsgUfaCfcCfuAfggaAfaUfaccagsasg 551 AD-63205 A-122745.13CfsusGfgUfaUfuUfCfCfuAfgGfgUfaCfaAfL96 495 A-126609.1usUfsgUfaccCfuaggaAfaUfaccAfgsasg 552 AD-63241 A-126589.2csusgguaUfuUfCfCfuaggGfuacaaL96 496 A-126611.3usUfsguaCfccUfaggaAfaUfaccagsasg 553 AD-63243 A-126621.3csusGfguaUfuUfCfCfuAfgGfguAfcaaL96 497 A-126624.1usUfsGfuaCfcCfuAfggaAfAfuaCfcAfgsasg 554 AD-63203 A-126593.1csusgguaUfuUfCfCfuaggGfuadCaaL96 498 A-122746.12usUfsgUfaCfcCfuAfggaAfaUfaCfcAfgsasg 555 AD-63223 A-122745.16CfsusGfgUfaUfuUfCfCfuAfgGfgUfaCfaAfL96 499 A-126612.1usUfsguaCfccuaggaAfaUfaccagsasg 556 AD-63231 A-126621.1csusGfguaUfuUfCfCfuAfgGfguAfcaaL96 500 A-126622.1usUfsGfuaCfcCfuAfggaAfaUfaCfcAfgsasg 557 AD-63199 A-122745.12CfsusGfgUfaUfuUfCfCfuAfgGfgUfaCfaAfL96 501 A-126608.1usUfsgUfaccCfuAfggaAfaUfaccAfgsasg 558 AD-63217 A-122745.15CfsusGfgUfaUfuUfCfCfuAfgGfgUfaCfaAfL96 502 A-126611.1usUfsguaCfccUfaggaAfaUfaccagsasg 559 AD-63229 A-122745.17CfsusGfgUfaUfuUfCfCfuAfgGfgUfaCfaAfL96 503 A-126613.1usUfsguaCfcCfUfaggaAfaUfaccagsasg 560 AD-63255 A-126621.5csusGfguaUfuUfCfCfuAfgGfguAfcaaL96 504 A-126626.1usUfsGfuAfCfcCfuAfggaAfAfuaCfcAfgsasg 561 AD-63226 A-126589.1csusgguaUfuUfCfCfuaggGfuacaaL96 505 A-122746.8usUfsgUfaCfcCfuAfggaAfaUfaCfcAfgsasg 562 AD-63211 A-122745.14CfsusGfgUfaUfuUfCfCfuAfgGfgUfaCfaAfL96 506 A-126610.1usUfsgUfacccuAfggaAfaUfaccAfgsasg 563 AD-63273 A-126621.8csusGfguaUfuUfCfCfuAfgGfguAfcaaL96 507 A-126629.1usUfsGfuaCfcCfuAfggaAfAfuAfccagsasg 564 AD-60940 A-122745.1CfsusGfgUfaUfuUfCfCfuAfgGfgUfaCfaAfL96 508 A-122746.1usUfsgUfaCfcCfuAfggaAfaUfaCfcAfgsasg 565 AD-63249 A-126621.4csusGfguaUfuUfCfCfuAfgGfguAfcaaL96 509 A-126625.1usUfsGfuAfCfcCfuAfggaAfAfuAfCfcAfgsasg 566 AD-63256 A-122745.19CfsusGfgUfaUfuUfCfCfuAfgGfgUfaCfaAfL96 510 A-126634.1usUfsgUfaccCfuAfggaAfaUfaCfcAfgsasg 567 AD-63280 A-126639.1csusGfgUfaUfuUfCfCfuAfgGfgUfaCfaAfL96 511 A-126587.3PusUfsgUfaCfcCfuAfggaAfaUfaCfcAfgsasg 568 AD-63237 A-126621.2csusGfguaUfuUfCfCfuAfgGfguAfcaaL96 512 A-126623.1usUfsGfuAfCfcCfuAfggaAfaUfaCfcAfgsasg 569 AD-63285 A-126621.10csusGfguaUfuUfCfCfuAfgGfguAfcaaL96 513 A-126631.1usUfsGfuaCfcCfuAfggaAfAfuAfccAfgsasg 570 AD-63215 A-126595.1csusgguaUfuUfCfdCuaggGfuacaaL96 514 A-122746.14usUfsgUfaCfcCfuAfggaAfaUfaCfcAfgsasg 571 AD-63222 A-122745.8CfsusGfgUfaUfuUfCfCfuAfgGfgUfaCfaAfL96 515 A-126604.1usUfsguaCfcCfuAfggaAfaUfaccAfgsasg 572 AD-63232 A-126590.1csusgguAfuuUfcCfUfagGfGfuacaaL96 516 A-122746.9usUfsgUfaCfcCfuAfggaAfaUfaCfcAfgsasg 573 AD-63218 A-126594.2csusgguaUfuUfCfCfuaggGfdTacaaL96 517 A-126611.7usUfsguaCfccUfaggaAfaUfaccagsasg 574 AD-63261 A-126621.6csusGfguaUfuUfCfCfuAfgGfguAfcaaL96 518 A-126627.1usUfsGfuaCfcCfuAfggaAfAfuAfCfcagsasg 575 AD-63267 A-126621.7csusGfguaUfuUfCfCfuAfgGfguAfcaaL96 519 A-126628.1usUfsGfuAfCfcCfuAfggaAfAfuAfCfcagsasg 576 AD-63234 A-122745.10CfsusGfgUfaUfuUfCfCfuAfgGfgUfaCfaAfL96 520 A-126606.1usUfsguaCfccuAfggaAfaUfaccAfgsasg 577 AD-63250 A-122745.18CfsusGfgUfaUfuUfCfCfuAfgGfgUfaCfaAfL96 521 A-126633.1ususgUfaCfcCfuAfggaAfaUfaCfcAfgsasg 578 AD-63212 A-126593.2csusgguaUfuUfCfCfuaggGfuadCaaL96 522 A-126611.6usUfsguaCfccUfaggaAfaUfaccagsasg 579 AD-63210 A-126602.1csusgguauuucdCuaggg(Tgn)acaaL96 523 A-122746.21usUfsgUfaCfcCfuAfggaAfaUfaCfcAfgsasg 580 AD-63244 A-126621.11csusGfguaUfuUfCfCfuAfgGfguAfcaaL96 524 A-126632.1usUfsGfuAfCfcCfuAfggaAfAfuAfccAfgsasg 581 AD-63235 A-126588.2csusgguAfuuuCfCfuAfggGfuacaaL96 525 A-126611.2usUfsguaCfccUfaggaAfaUfaccagsasg 582 AD-63279 A-126621.9csusGfguaUfuUfCfCfuAfgGfguAfcaaL96 526 A-126630.1usUfsGfuAfCfcCfuAfggaAfAfuAfccagsasg 583 AD-63227 A-126597.1csusgguAfuuucCfuagggdTacaaL96 527 A-122746.16usUfsgUfaCfcCfuAfggaAfaUfaCfcAfgsasg 584 AD-63220 A-126588.1csusgguAfuuuCfCfuAfggGfuacaaL96 528 A-122746.7usUfsgUfaCfcCfuAfggaAfaUfaCfcAfgsasg 585 AD-63238 A-126591.1csusgguAfuuucCfuaggguacaaL96 529 A-122746.10usUfsgUfaCfcCfuAfggaAfaUfaCfcAfgsasg 586 AD-63242 A-126598.2csusgguAfuuucCfdTaggguacaaL96 530 A-126611.11usUfsguaCfccUfaggaAfaUfaccagsasg 587 AD-63239 A-126599.1csusgguauuucCfdTaggguacaaL96 531 A-122746.18usUfsgUfaCfcCfuAfggaAfaUfaCfcAfgsasg 588 AD-63233 A-126598.1csusgguAfuuucCfdTaggguacaaL96 532 A-122746.17usUfsgUfaCfcCfuAfggaAfaUfaCfcAfgsasg 589 AD-63268 A-126636.1CfsusGfgUfaUfuUfCfcuAfgGfgUfaCfaAfL96 533 A-122746.22usUfsgUfaCfcCfuAfggaAfaUfaCfcAfgsasg 590 AD-63221 A-126596.1csusgguAfuuucCfuaggguadCaaL96 534 A-122746.15usUfsgUfaCfcCfuAfggaAfaUfaCfcAfgsasg 591 AD-63236 A-126597.2csusgguAfuuucCfuagggdTacaaL96 535 A-126611.10usUfsguaCfccUfaggaAfaUfaccagsasg 592 AD-63197 A-126592.1csusgguauuucCfUfaggguacaaL96 536 A-122746.11usUfsgUfaCfcCfuAfggaAfaUfaCfcAfgsasg 593 AD-63224 A-126595.2csusgguaUfuUfCfdCuaggGfuacaaL96 537 A-126611.8usUfsguaCfccUfaggaAfaUfaccagsasg 594 AD-63200 A-126590.2csusgguAfuuUfcCfUfagGfGfuacaaL96 538 A-126611.4usUfsguaCfccUfaggaAfaUfaccagsasg 595 AD-63262 A-122745.20CfsusGfgUfaUfuUfCfCfuAfgGfgUfaCfaAfL96 539 A-126635.1usUfsgUfaCfcCfuAfggaaaUfaCfcAfgsasg 596 AD-63204 A-126601.1csusgguauuucdCuaggguacaaL96 540 A-122746.20usUfsgUfaCfcCfuAfggaAfaUfaCfcAfgsasg 597 AD-63230 A-126596.2csusgguAfuuucCfuaggguadCaaL96 541 A-126611.9usUfsguaCfccUfaggaAfaUfaccagsasg 598 AD-63198 A-126600.1csusgguauuucdCdTaggguacaaL96 542 A-122746.19usUfsgUfaCfcCfuAfggaAfaUfaCfcAfgsasg 599 AD-63206 A-126591.2csusgguAfuuucCfuaggguacaaL96 543 A-126611.5usUfsguaCfccUfaggaAfaUfaccagsasg 600

TABLE 9 TMPRSS6 Single Dose Screen 10 nM 0.1 nM DuplexID Avg AvgAD-63214 12.40 19.46 AD-63240 12.29 27.03 AD-63209 17.11 23.38 AD-6320814.77 23.31 AD-63202 14.87 27.08 AD-63216 15.97 34.05 AD-63219 18.4727.82 AD-63228 19.44 34.52 AD-63205 15.44 38.23 AD-63241 18.81 41.42AD-63243 19.15 30.87 AD-63203 17.06 42.12 AD-63223 21.98 27.52 AD-6323122.42 30.68 AD-63199 17.74 39.50 AD-63217 18.81 38.99 AD-63229 22.3333.42 AD-63255 21.06 34.31 AD-63226 18.36 41.65 AD-63211 26.00 32.07AD-63273 23.11 34.96 AD-60940 22.99 34.34 AD-63249 30.83 28.35 AD-6325623.18 35.19 AD-63280 25.10 32.42 AD-63237 23.95 35.43 AD-63285 21.5339.60 AD-63215 29.27 42.54 AD-63222 23.88 38.24 AD-63232 30.29 35.04AD-63218 27.02 37.31 AD-63261 24.22 46.61 AD-63267 28.32 38.90 AD-6323424.42 55.83 AD-63250 26.77 47.92 AD-63212 28.43 46.01 AD-63210 27.9144.35 AD-63244 30.66 45.65 AD-63235 32.75 51.82 AD-63279 38.00 48.80AD-63227 33.15 58.12 AD-63220 38.31 54.08 AD-63238 45.56 51.50 AD-6324247.96 54.26 AD-63239 51.98 49.22 AD-63233 51.37 65.83 AD-63268 41.2282.16 AD-63221 57.02 65.11 AD-63236 49.86 71.66 AD-63197 47.67 78.29AD-63224 67.73 60.88 AD-63200 62.89 67.68 AD-63262 64.25 79.72 AD-6320468.01 80.99 AD-63230 66.88 81.04 AD-63198 65.67 78.28 AD-63206 65.1082.71

TABLE 10 TMPRS S6 Modified Sequences SEQ SEQ ID ID DuplexID SenseIDSense Sequence NO: AntisenseID Antisense Sequence NO: AD-63214A-126586.2 Y44CfsusGfgUfaUfuUfCfCfuAfgGfgUfaCfaAfL96 601 A-126587.2PusUfsgUfaCfcCfuAfggaAfaUfaCfcAfgsasg 658 AD-63240 A-122745.11CfsusGfgUfaUfuUfCfCfuAfgGfgUfaCfaAfL96 602 A-126607.1usUfsguaCfcCfuAfggaAfaUfaccagsasg 659 AD-63209 A-126594.1csusgguaUfuUfCfCfuaggGfdTacaaL96 603 A-122746.13usUfsgUfaCfcCfuAfggaAfaUfaCfcAfgsasg 660 AD-63208 A-122745.6CfsusGfgUfaUfuUfCfCfuAfgGfgUfaCfaAfL96 604 A-126587.1PusUfsgUfaCfcCfuAfggaAfaUfaCfcAfgsasg 661 AD-63202 A-126586.1Y44CfsusGfgUfaUfuUfCfCfuAfgGfgUfaCfaAfL96 605 A-122746.6usUfsgUfaCfcCfuAfggaAfaUfaCfcAfgsasg 662 AD-63216 A-122745.7CfsusGfgUfaUfuUfCfCfuAfgGfgUfaCfaAfL96 606 A-126603.1usUfsgUfaCfccuAfggaAfaUfaCfcAfgsasg 663 AD-63219 A-126617.1gsgsUfaUfuUfCfCfuAfgGfgUfaCfaAfL96 607 A-126618.1PusUfsgUfaCfcCfuAfggaAfaUfaCfcsasg 664 AD-63228 A-122745.9CfsusGfgUfaUfuUfCfCfuAfgGfgUfaCfaAfL96 608 A-126605.1usUfsgUfaCfcCfuAfggaAfaUfaccagsasg 665 AD-63205 A-122745.13CfsusGfgUfaUfuUfCfCfuAfgGfgUfaCfaAfL96 609 A-126609.1usUfsgUfaccCfuaggaAfaUfaccAfgsasg 666 AD-63241 A-126589.2csusgguaUfuUfCfCfuaggGfuacaaL96 610 A-126611.3usUfsguaCfccUfaggaAfaUfaccagsasg 667 AD-63243 A-126621.3csusGfguaUfuUfCfCfuAfgGfguAfcaaL96 611 A-126624.1usUfsGfuaCfcCfuAfggaAfAfuaCfcAfgsasg 668 AD-63203 A-126593.1csusgguaUfuUfCfCfuaggGfuadCaaL96 612 A-122746.12usUfsgUfaCfcCfuAfggaAfaUfaCfcAfgsasg 669 AD-63223 A-122745.16CfsusGfgUfaUfuUfCfCfuAfgGfgUfaCfaAfL96 613 A-126612.1usUfsguaCfccuaggaAfaUfaccagsasg 670 AD-63231 A-126621.1csusGfguaUfuUfCfCfuAfgGfguAfcaaL96 614 A-126622.1usUfsGfuaCfcCfuAfggaAfaUfaCfcAfgsasg 671 AD-63199 A-122745.12CfsusGfgUfaUfuUfCfCfuAfgGfgUfaCfaAfL96 615 A-126608.1usUfsgUfaccCfuAfggaAfaUfaccAfgsasg 672 AD-63217 A-122745.15CfsusGfgUfaUfuUfCfCfuAfgGfgUfaCfaAfL96 616 A-126611.1usUfsguaCfccUfaggaAfaUfaccagsasg 673 AD-63229 A-122745.17CfsusGfgUfaUfuUfCfCfuAfgGfgUfaCfaAfL96 617 A-126613.1usUfsguaCfcCfUfaggaAfaUfaccagsasg 674 AD-63255 A-126621.5csusGfguaUfuUfCfCfuAfgGfguAfcaaL96 618 A-126626.1usUfsGfuAfCfcCfuAfggaAfAfuaCfcAfgsasg 675 AD-63226 A-126589.1csusgguaUfuUfCfCfuaggGfuacaaL96 619 A-122746.8usUfsgUfaCfcCfuAfggaAfaUfaCfcAfgsasg 676 AD-63211 A-122745.14CfsusGfgUfaUfuUfCfCfuAfgGfgUfaCfaAfL96 620 A-126610.1usUfsgUfacccuAfggaAfaUfaccAfgsasg 677 AD-63273 A-126621.8csusGfguaUfuUfCfCfuAfgGfguAfcaaL96 621 A-126629.1usUfsGfuaCfcCfuAfggaAfAfuAfccagsasg 678 AD-60940 A-122745.1CfsusGfgUfaUfuUfCfCfuAfgGfgUfaCfaAfL96 622 A-122746.1usUfsgUfaCfcCfuAfggaAfaUfaCfcAfgsasg 679 AD-63249 A-126621.4csusGfguaUfuUfCfCfuAfgGfguAfcaaL96 623 A-126625.1usUfsGfuAfCfcCfuAfggaAfAfuAfCfcAfgsasg 680 AD-63256 A-122745.19CfsusGfgUfaUfuUfCfCfuAfgGfgUfaCfaAfL96 624 A-126634.1usUfsgUfaccCfuAfggaAfaUfaCfcAfgsasg 681 AD-63280 A-126639.1csusGfgUfaUfuUfCfCfuAfgGfgUfaCfaAfL96 625 A-126587.3PusUfsgUfaCfcCfuAfggaAfaUfaCfcAfgsasg 682 AD-63237 A-126621.2csusGfguaUfuUfCfCfuAfgGfguAfcaaL96 626 A-126623.1usUfsGfuAfCfcCfuAfggaAfaUfaCfcAfgsasg 683 AD-63285 A-126621.10csusGfguaUfuUfCfCfuAfgGfguAfcaaL96 627 A-126631.1usUfsGfuaCfcCfuAfggaAfAfuAfccAfgsasg 684 AD-63215 A-126595.1csusgguaUfuUfCfdCuaggGfuacaaL96 628 A-122746.14usUfsgUfaCfcCfuAfggaAfaUfaCfcAfgsasg 685 AD-63222 A-122745.8CfsusGfgUfaUfuUfCfCfuAfgGfgUfaCfaAfL96 629 A-126604.1usUfsguaCfcCfuAfggaAfaUfaccAfgsasg 686 AD-63232 A-126590.1csusgguAfuuUfcCfUfagGfGfuacaaL96 630 A-122746.9usUfsgUfaCfcCfuAfggaAfaUfaCfcAfgsasg 687 AD-63218 A-126594.2csusgguaUfuUfCfCfuaggGfdTacaaL96 631 A-126611.7usUfsguaCfccUfaggaAfaUfaccagsasg 688 AD-63261 A-126621.6csusGfguaUfuUfCfCfuAfgGfguAfcaaL96 632 A-126627.1usUfsGfuaCfcCfuAfggaAfAfuAfCfcagsasg 689 AD-63267 A-126621.7csusGfguaUfuUfCfCfuAfgGfguAfcaaL96 633 A-126628.1usUfsGfuAfCfcCfuAfggaAfAfuAfCfcagsasg 690 AD-63234 A-122745.10CfsusGfgUfaUfuUfCfCfuAfgGfgUfaCfaAfL96 634 A-126606.1usUfsguaCfccuAfggaAfaUfaccAfgsasg 691 AD-63250 A-122745.18CfsusGfgUfaUfuUfCfCfuAfgGfgUfaCfaAfL96 635 A-126633.1ususgUfaCfcCfuAfggaAfaUfaCfcAfgsasg 692 AD-63212 A-126593.2csusgguaUfuUfCfCfuaggGfuadCaaL96 636 A-126611.6usUfsguaCfccUfaggaAfaUfaccagsasg 693 AD-63210 A-126602.1csusgguauuucdCuaggg(Tgn)acaaL96 637 A-122746.21usUfsgUfaCfcCfuAfggaAfaUfaCfcAfgsasg 694 AD-63244 A-126621.11csusGfguaUfuUfCfCfuAfgGfguAfcaaL96 638 A-126632.1usUfsGfuAfCfcCfuAfggaAfAfuAfccAfgsasg 695 AD-63235 A-126588.2csusgguAfuuuCfCfuAfggGfuacaaL96 639 A-126611.2usUfsguaCfccUfaggaAfaUfaccagsasg 696 AD-63279 A-126621.9csusGfguaUfuUfCfCfuAfgGfguAfcaaL96 640 A-126630.1usUfsGfuAfCfcCfuAfggaAfAfuAfccagsasg 697 AD-63227 A-126597.1csusgguAfuuucCfuagggdTacaaL96 641 A-122746.16usUfsgUfaCfcCfuAfggaAfaUfaCfcAfgsasg 698 AD-63220 A-126588.1csusgguAfuuuCfCfuAfggGfuacaaL96 642 A-122746.7usUfsgUfaCfcCfuAfggaAfaUfaCfcAfgsasg 699 AD-63238 A-126591.1csusgguAfuuucCfuaggguacaaL96 643 A-122746.10usUfsgUfaCfcCfuAfggaAfaUfaCfcAfgsasg 700 AD-63242 A-126598.2csusgguAfuuucCfdTaggguacaaL96 644 A-126611.11usUfsguaCfccUfaggaAfaUfaccagsasg 701 AD-63239 A-126599.1csusgguauuucCfdTaggguacaaL96 645 A-122746.18usUfsgUfaCfcCfuAfggaAfaUfaCfcAfgsasg 702 AD-63233 A-126598.1csusgguAfuuucCfdTaggguacaaL96 646 A-122746.17usUfsgUfaCfcCfuAfggaAfaUfaCfcAfgsasg 703 AD-63268 A-126636.1CfsusGfgUfaUfuUfCfcuAfgGfgUfaCfaAfL96 647 A-122746.22usUfsgUfaCfcCfuAfggaAfaUfaCfcAfgsasg 704 AD-63221 A-126596.1csusgguAfuuucCfuaggguadCaaL96 648 A-122746.15usUfsgUfaCfcCfuAfggaAfaUfaCfcAfgsasg 705 AD-63236 A-126597.2csusgguAfuuucCfuagggdTacaaL96 649 A-126611.10usUfsguaCfccUfaggaAfaUfaccagsasg 706 AD-63197 A-126592.1csusgguauuucCfUfaggguacaaL96 650 A-122746.11usUfsgUfaCfcCfuAfggaAfaUfaCfcAfgsasg 707 AD-63224 A-126595.2csusgguaUfuUfCfdCuaggGfuacaaL96 651 A-126611.8usUfsguaCfccUfaggaAfaUfaccagsasg 708 AD-63200 A-126590.2csusgguAfuuUfcCfUfagGfGfuacaaL96 652 A-126611.4usUfsguaCfccUfaggaAfaUfaccagsasg 709 AD-63262 A-122745.20CfsusGfgUfaUfuUfCfCfuAfgGfgUfaCfaAfL96 653 A-126635.1usUfsgUfaCfcCfuAfggaaaUfaCfcAfgsasg 710 AD-63204 A-126601.1csusgguauuucdCuaggguacaaL96 654 A-122746.20usUfsgUfaCfcCfuAfggaAfaUfaCfcAfgsasg 711 AD-63230 A-126596.2csusgguAfuuucCfuaggguadCaaL96 655 A-126611.9usUfsguaCfccUfaggaAfaUfaccagsasg 712 AD-63198 A-126600.1csusgguauuucdCdTaggguacaaL96 656 A-122746.19usUfsgUfaCfcCfuAfggaAfaUfaCfcAfgsasg 713 AD-63206 A-126591.2csusgguAfuuucCfuaggguacaaL96 657 A-126611.5usUfsguaCfccUfaggaAfaUfaccagsasg 714

TABLE 11 TMPRSS6 Single Dose Screen 10 nM 0.1 nM DuplexID Avg SD Avg SDAD-60998 26.1 3.1 42.9 13.3 AD-60970 24.3 9.3 39.0 24.2 AD-61002 27.58.5 32.1 9.8 AD-60994 19.9 5.8 28.2 9.3 AD-60992 57.9 15.4 67.5 13.6AD-61006 25.8 2.5 33.4 8.7 AD-59743 21.1 3.2 31.7 8.1 AD-60966 64.6 15.676.0 18.2 AD-60952 44.1 10.7 76.9 16.5 AD-61000 37.2 5.8 43.3 12.7AD-60949 94.9 22.3 91.3 13.2 AD-60969 100.7 18.5 124.5 43.0 AD-6096793.7 6.4 112.1 31.5 AD-60984 44.7 21.4 58.2 9.6 AD-60943 65.6 11.0 61.79.8 AD-61001 69.2 8.3 100.8 8.4 AD-60986 38.9 13.9 58.9 4.8 AD-6098861.7 12.0 68.6 15.2 AD-60993 92.1 13.1 86.5 10.0 AD-60987 113.9 15.397.9 21.0 AD-60997 54.8 7.2 75.8 16.4 AD-60973 61.5 15.7 80.8 9.3AD-61005 116.8 23.4 128.1 10.8 AD-60985 71.2 15.1 78.7 14.6 AD-61003101.0 15.2 97.5 15.8 AD-60989 75.8 9.8 97.2 20.8 AD-60955 108.6 23.4102.0 16.6 AD-60991 96.6 19.4 95.6 12.4 AD-61004 111.1 6.4 110.9 18.3AD-60961 96.9 36.0 84.1 28.2 AD-60999 106.7 12.7 92.3 24.6 AD-60990 92.938.4 97.6 16.8 AD-60996 71.2 7.5 101.5 8.9

Example 13. Optimization of AD-60940

Additional duplexes targeting TMPRSS6 were produced and screened invitro for efficacy using the materials and methods below.

Design, Synthesis, and In Vitro Screening of Additional siRNAssiRNA Design

TMPRSS6 duplexes, 19 nucleotides long for both the sense and antisensestrand, were designed using the human TMPRSS6 mRNA sequence set forth inGenBank Accession No. NM_153609.3. Three thousand one hundred and eightyduplexes were initially identified that did not contain repeats longerthan 7 nucleotides, spanning substantially the entire 3209 nucleotidetranscript. All 3180 duplexes were then scored for predicted efficacyaccording to a linear model that evaluates the nucleotide pair at eachduplex position, and the dose and cell line used for screening. Theduplexes were also matched against all transcripts in the human RefSeqcollection using a custom brute force algorithm, and scored for lowestnumbers of mismatches (per strand) to transcripts other than TMPRSS6.Duplexes to be synthesized and screened were then selected from the3180, according to the following scheme: Beginning at the 5′ end of thetranscript, a duplex was selected within a “window” of every 10±2nucleotides that had the highest predicted efficacy, had at least onemismatch in both strands to all transcripts other than TMPRSS6, and hadnot already been synthesized and screened as part of other duplex sets.If no duplex is identified within a given window that satisfied allcriteria, that window was skipped. Three hundred and three duplexes wereselected according to the above criteria. An additional 31 duplexes werealso selected.

A detailed list of the 334 TMPRSS6 sense and antisense strand sequencesis shown in Table 12.

Cell Culture and Transfections

Hep3B2.1-7 cells were obtained from American Type Culture Collection(Rockville, Md., cat. No. HB-8064) and cultured in EMEM (ATCC #30-2003),supplemented to contain 10% fetal calf serum (FCS) (Biochrom AG, Berlin,Germany, cat. No. S0115) and Penicillin 100 U/ml, Streptomycin 100 mg/ml(Biochrom AG, Berlin, Germany, cat. No. A2213), at 37° C. in anatmosphere with 5% CO₂ in a humidified incubator (Heraeus HERAcell,Kendro Laboratory Products, Langenselbold, Germany).

Transfection of dsRNA was performed directly after seeding 15,000cells/well on a 96-well plate, and was carried out with Lipofectamine2000 (Invitrogen GmbH, Karlsruhe, Germany, cat.No. 11668-019) asdescribed by the manufacturer. Transfections were performed inquadruplicates and dsRNAs were transfected at a concentration of 10 nM.

Branched DNA Assays—QunatiGene 2.0 (Panomics cat #: QS0011)

For measurement of TMPRSS6 mRNA cells were harvested 24 hours aftertransfection and lysed at 53° C. following procedures recommended by themanufacturer of the Quantigene II Kit for TMPRSS6 and Quantigene IExplore Kit for bDNA (Panomics, Fremont, Calif., USA, cat. No. 15735 orQG0004, respectively). Subsequently, 50 μl of the lysates were incubatedwith probesets specific to human TMPRSS6 and 10 μl of the lysates forhuman GAPDH and processed according to the manufacturer's protocol forQuantiGene. Chemoluminescence was measured in a Victor2-Light (PerkinElmer, Wiesbaden, Germany) as RLUs (relative light units) and valuesobtained with the human TMPRSS6 probeset were normalized to therespective human GAPDH values for each well and then related to the meanof three unrelated control dsRNAs.

The in vitro efficacy of the compounds is shown in Table 13.

TABLE 12 Additional modified TMPRSS6 siRNAs SEQ Position in SEQ IDDuplex ID Sense Sequence ID NO: Sense ID NM_153609.3 Antisense SequenceNO: Antisense ID AD-63290.1 UGAGCCAGACCCAGUCCAGdTdT  715 A-126858.1   3-21 CUGGACUGGGUCUGGCUCAdTdT 1049 A-126859.1 AD-63296.1GACCCAGUCCAGCUCUGGUdTdT  716 A-126860.1   10-28 ACCAGAGCUGGACUGGGUCdTdT1050 A-126861.1 AD-63302.1 CUCUGGUGCCUGCCCUCUGdTdT  717 A-126862.1  22-40 CAGAGGGCAGGCACCAGAGdTdT 1051 A-126863.1 AD-63308.1GCCCUCUGGUGCGAGCUGAdTdT  718 A-126864.1   33-51 UCAGCUCGCACCAGAGGGCdTdT1052 A-126865.1 AD-63314.1 GGUGCGAGCUGACCUGAGAdTdT  719 A-126866.1  40-58 UCUCAGGUCAGCUCGCACCdTdT 1053 A-126867.1 AD-63320.1UGACCUGAGAUGCACUUCCdTdT  720 A-126868.1   49-67 GGAAGUGCAUCUCAGGUCAdTdT1054 A-126869.1 AD-63326.1 UGCACUUCCCUCCUCUGUGdTdT  721 A-126870.1  59-77 CACAGAGGAGGGAAGUGCAdTdT 1055 A-126871.1 AD-63332.1CUGUGAGCUGUCUCGGCACdTdT  722 A-126872.1   73-91 GUGCCGAGACAGCUCACAGdTdT1056 A-126873.1 AD-63291.1 GUCUCGGCACCCACUUGCAdTdT  723 A-126874.1  82-100 UGCAAGUGGGUGCCGAGACdTdT 1057 A-126875.1 AD-63297.1CCACUUGCAGUCACUGCCGdTdT  724 A-126876.1   92-110 CGGCAGUGACUGCAAGUGGdTdT1058 A-126877.1 AD-63303.1 GUCACUGCCGCCUGAUGUUdTdT  725 A-126878.1 101-119 AACAUCAGGCGGCAGUGACdTdT 1059 A-126879.1 AD-63309.1GCCUGAUGUUGUUACUCUUdTdT  726 A-126880.1  110-128 AAGAGUAACAACAUCAGGCdTdT1060 A-126881.1 AD-63315.1 UUACUCUUCCACUCCAAAAdTdT  727 A-126882.1 121-139 UUUUGGAGUGGAAGAGUAAdTdT 1061 A-126883.1 AD-63321.1ACUCCAAAAGGAUGCCCGUdTdT  728 A-126884.1  131-149 ACGGGCAUCCUUUUGGAGUdTdT1062 A-126885.1 AD-63327.1 UGCCCGUGGCCGAGGCCCCdTdT  729 A-126886.1 143-161 GGGGCCUCGGCCACGGGCAdTdT 1063 A-126887.1 AD-63333.1UGGCCGAGGCCCCCCAGGUdTdT  730 A-126888.1  149-167 ACCUGGGGGGCCUCGGCCAdTdT1064 A-126889.1 AD-63292.1 CCAGGUGGCUGGCGGGCAGdTdT  731 A-126890.1 162-180 CUGCCCGCCAGCCACCUGGdTdT 1065 A-126891.1 AD-63298.1GCGGGCAGGGGGACGGAGGdTdT  732 A-126892.1  173-191 CCUCCGUCCCCCUGCCCGCdTdT1066 A-126893.1 AD-63304.1 GGACGGAGGUGAUGGCGAGdTdT  733 A-126894.1 183-201 CUCGCCAUCACCUCCGUCCdTdT 1067 A-126895.1 AD-63310.1GUGAUGGCGAGGAAGCGGAdTdT  734 A-126896.1  191-209 UCCGCUUCCUCGCCAUCACdTdT1068 A-126897.1 AD-63316.1 GAAGCGGAGCCGGAGGGGAdTdT  735 A-126898.1 202-220 UCCCCUCCGGCUCCGCUUCdTdT 1069 A-126899.1 AD-63322.1GCCGGAGGGGAUGUUCAAGdTdT  736 A-126900.1  210-228 CUUGAACAUCCCCUCCGGCdTdT1070 A-126901.1 AD-63328.1 UGUUCAAGGCCUGUGAGGAdTdT  737 A-126902.1 221-239 UCCUCACAGGCCUUGAACAdTdT 1071 A-126903.1 AD-63334.1CUGUGAGGACUCCAAGAGAdTdT  738 A-126904.1  231-249 UCUCUUGGAGUCCUCACAGdTdT1072 A-126905.1 AD-63293.1 ACUCCAAGAGAAAAGCCCGdTdT  739 A-126906.1 239-257 CGGGCUUUUCUCUUGGAGUdTdT 1073 A-126907.1 AD-63299.1GCCCGGGGCUACCUCCGCCdTdT  740 A-126908.1  253-271 GGCGGAGGUAGCCCCGGGCdTdT1074 A-126909.1 AD-63305.1 ACCUCCGCCUGGUGCCCCUdTdT  741 A-126910.1 263-281 AGGGGCACCAGGCGGAGGUdTdT 1075 A-126911.1 AD-63311.1GCCUGGUGCCCCUGUUUGUdTdT  742 A-126912.1  269-287 ACAAACAGGGGCACCAGGCdTdT1076 A-126913.1 AD-63317.1 UGUUUGUGCUGCUGGCCCUdTdT  743 A-126914.1 281-299 AGGGCCAGCAGCACAAACAdTdT 1077 A-126915.1 AD-63323.1UGCUGGCCCUGCUCGUGCUdTdT  744 A-126916.1  290-308 AGCACGAGCAGGGCCAGCAdTdT1078 A-126917.1 AD-63329.1 GCUCGUGCUGGCUUCGGCGdTdT  745 A-126918.1 300-318 CGCCGAAGCCAGCACGAGCdTdT 1079 A-126919.1 AD-63335.1UCGGCGGGGGUGCUACUCUdTdT  746 A-126920.1  313-331 AGAGUAGCACCCCCGCCGAdTdT1080 A-126921.1 AD-63294.1 CGGCGGGGGUGCUACUCUGdTdT  747 A-126922.1 314-332 CAGAGUAGCACCCCCGCCGdTdT 1081 A-126923.1 AD-63300.1GGCGGGGGUGCUACUCUGGdTdT  748 A-126924.1  315-333 CCAGAGUAGCACCCCCGCCdTdT1082 A-126925.1 AD-63306.1 GCGGGGGUGCUACUCUGGUdTdT  749 A-126926.1 316-334 ACCAGAGUAGCACCCCCGCdTdT 1083 A-126927.1 AD-63312.1CGGGGGUGCUACUCUGGUAdTdT  750 A-126928.1  317-335 UACCAGAGUAGCACCCCCGdTdT1084 A-126929.1 AD-63318.1 GGGGGUGCUACUCUGGUAUdTdT  751 A-126930.1 318-336 AUACCAGAGUAGCACCCCCdTdT 1085 A-126931.1 AD-63324.1GGGUGCUACUCUGGUAUUUdTdT  752 A-126932.1  320-338 AAAUACCAGAGUAGCACCCdTdT1086 A-126933.1 AD-63330.1 GGUGCUACUCUGGUAUUUCdTdT  753 A-126934.1 321-339 GAAAUACCAGAGUAGCACCdTdT 1087 A-126935.1 AD-63336.1GUGCUACUCUGGUAUUUCCdTdT  754 A-126936.1  322-340 GGAAAUACCAGAGUAGCACdTdT1088 A-126937.1 AD-63295.1 GCUACUCUGGUAUUUCCUAdTdT  755 A-126938.1 324-342 UAGGAAAUACCAGAGUAGCdTdT 1089 A-126939.1 AD-63301.1CUACUCUGGUAUUUCCUAGdTdT  756 A-126940.1  325-343 CUAGGAAAUACCAGAGUAGdTdT1090 A-126941.1 AD-63307.1 UACUCUGGUAUUUCCUAGGdTdT  757 A-126942.1 326-344 CCUAGGAAAUACCAGAGUAdTdT 1091 A-126943.1 AD-63313.1ACUCUGGUAUUUCCUAGGGdTdT  758 A-126944.1  327-345 CCCUAGGAAAUACCAGAGUdTdT1092 A-126945.1 AD-63319.1 CUCUGGUAUUUCCUAGGGUdTdT  759 A-126946.1 328-346 ACCCUAGGAAAUACCAGAGdTdT 1093 A-126947.1 AD-63325.1CUGGUAUUUCCUAGGGUACdTdT  760 A-126948.1  330-348 GUACCCUAGGAAAUACCAGdTdT1094 A-126949.1 AD-63331.1 GUAUUUCCUAGGGUACAAGdTdT  761 A-126950.1 333-351 CUUGUACCCUAGGAAAUACdTdT 1095 A-126951.1 AD-63337.1UAUUUCCUAGGGUACAAGGdTdT  762 A-126952.1  334-352 CCUUGUACCCUAGGAAAUAdTdT1096 A-126953.1 AD-63343.1 AUUUCCUAGGGUACAAGGCdTdT  763 A-126954.1 335-353 GCCUUGUACCCUAGGAAAUdTdT 1097 A-126955.1 AD-63349.1UUUCCUAGGGUACAAGGCGdTdT  764 A-126956.1  336-354 CGCCUUGUACCCUAGGAAAdTdT1098 A-126957.1 AD-63355.1 UUCCUAGGGUACAAGGCGGdTdT  765 A-126958.1 337-355 CCGCCUUGUACCCUAGGAAdTdT 1099 A-126959.1 AD-63361.1CCUAGGGUACAAGGCGGAGdTdT  766 A-126960.1  339-357 CUCCGCCUUGUACCCUAGGdTdT1100 A-126961.1 AD-63367.1 CUAGGGUACAAGGCGGAGGdTdT  767 A-126962.1 340-358 CCUCCGCCUUGUACCCUAGdTdT 1101 A-126963.1 AD-63373.1UAGGGUACAAGGCGGAGGUdTdT  768 A-126964.1  341-359 ACCUCCGCCUUGUACCCUAdTdT1102 A-126965.1 AD-63379.1 AGGGUACAAGGCGGAGGUGdTdT  769 A-126966.1 342-360 CACCUCCGCCUUGUACCCUdTdT 1103 A-126967.1 AD-63338.1GGGUACAAGGCGGAGGUGAdTdT  770 A-126968.1  343-361 UCACCUCCGCCUUGUACCCdTdT1104 A-126969.1 AD-63344.1 GGUACAAGGCGGAGGUGAUdTdT  771 A-126970.1 344-362 AUCACCUCCGCCUUGUACCdTdT 1105 A-126971.1 AD-63350.1GUACAAGGCGGAGGUGAUGdTdT  772 A-126972.1  345-363 CAUCACCUCCGCCUUGUACdTdT1106 A-126973.1 AD-63356.1 UACAAGGCGGAGGUGAUGGdTdT  773 A-126974.1 346-364 CCAUCACCUCCGCCUUGUAdTdT 1107 A-126975.1 AD-63362.1ACAAGGCGGAGGUGAUGGUdTdT  774 A-126976.1  347-365 ACCAUCACCUCCGCCUUGUdTdT1108 A-126977.1 AD-63368.1 CAAGGCGGAGGUGAUGGUCdTdT  775 A-126978.1 348-366 GACCAUCACCUCCGCCUUGdTdT 1109 A-126979.1 AD-63374.1AAGGCGGAGGUGAUGGUCAdTdT  776 A-126980.1  349-367 UGACCAUCACCUCCGCCUUdTdT1110 A-126981.1 AD-63380.1 AGGCGGAGGUGAUGGUCAGdTdT  777 A-126982.1 350-368 CUGACCAUCACCUCCGCCUdTdT 1111 A-126983.1 AD-63339.1UGAUGGUCAGCCAGGUGUAdTdT  778 A-126984.1  359-377 UACACCUGGCUGACCAUCAdTdT1112 A-126985.1 AD-63345.1 CCAGGUGUACUCAGGCAGUdTdT  779 A-126986.1 369-387 ACUGCCUGAGUACACCUGGdTdT 1113 A-126987.1 AD-63351.1GCAGUCUGCGUGUACUCAAdTdT  780 A-126988.1  383-401 UUGAGUACACGCAGACUGCdTdT1114 A-126989.1 AD-63357.1 GCGUGUACUCAAUCGCCACdTdT  781 A-126990.1 390-408 GUGGCGAUUGAGUACACGCdTdT 1115 A-126991.1 AD-63363.1UCGCCACUUCUCCCAGGAUdTdT  782 A-126992.1  402-420 AUCCUGGGAGAAGUGGCGAdTdT1116 A-126993.1 AD-63369.1 CUCCCAGGAUCUUACCCGCdTdT  783 A-126994.1 411-429 GCGGGUAAGAUCCUGGGAGdTdT 1117 A-126995.1 AD-63375.1UACCCGCCGGGAAUCUAGUdTdT  784 A-126996.1  423-441 ACUAGAUUCCCGGCGGGUAdTdT1118 A-126997.1 AD-63381.1 CCGGGAAUCUAGUGCCUUCdTdT  785 A-126998.1 429-447 GAAGGCACUAGAUUCCCGGdTdT 1119 A-126999.1 AD-63340.1AGUGCCUUCCGCAGUGAAAdTdT  786 A-127000.1  439-457 UUUCACUGCGGAAGGCACUdTdT1120 A-127001.1 AD-63346.1 GUGAAACCGCCAAAGCCCAdTdT  787 A-127002.1 452-470 UGGGCUUUGGCGGUUUCACdTdT 1121 A-127003.1 AD-63352.1CGCCAAAGCCCAGAAGAUGdTdT  788 A-127004.1  459-477 CAUCUUCUGGGCUUUGGCGdTdT1122 A-127005.1 AD-63358.1 CAGAAGAUGCUCAAGGAGCdTdT  789 A-127006.1 469-487 GCUCCUUGAGCAUCUUCUGdTdT 1123 A-127007.1 AD-63364.1UCAAGGAGCUCAUCACCAGdTdT  790 A-127008.1  479-497 CUGGUGAUGAGCUCCUUGAdTdT1124 A-127009.1 AD-63370.1 ACCAGCACCCGCCUGGGAAdTdT  791 A-127010.1 493-511 UUCCCAGGCGGGUGCUGGUdTdT 1125 A-127011.1 AD-63376.1GCCUGGGAACUUACUACAAdTdT  792 A-127012.1  503-521 UUGUAGUAAGUUCCCAGGCdTdT1126 A-127013.1 AD-63382.1 GAACUUACUACAACUCCAGdTdT  793 A-127014.1 509-527 CUGGAGUUGUAGUAAGUUCdTdT 1127 A-127015.1 AD-63341.1AACUCCAGCUCCGUCUAUUdTdT  794 A-127016.1  520-538 AAUAGACGGAGCUGGAGUUdTdT1128 A-127017.1 AD-63347.1 CCGUCUAUUCCUUUGGGGAdTdT  795 A-127018.1 530-548 UCCCCAAAGGAAUAGACGGdTdT 1129 A-127019.1 AD-63353.1UUGGGGAGGGACCCCUCACdTdT  796 A-127020.1  542-560 GUGAGGGGUCCCUCCCCAAdTdT1130 A-127021.1 AD-63359.1 CCCCUCACCUGCUUCUUCUdTdT  797 A-127022.1 553-571 AGAAGAAGCAGGUGAGGGGdTdT 1131 A-127023.1 AD-63365.1CUGCUUCUUCUGGUUCAUUdTdT  798 A-127024.1  561-579 AAUGAACCAGAAGAAGCAGdTdT1132 A-127025.1 AD-63371.1 CUGGUUCAUUCUCCAAAUCdTdT  799 A-127026.1 570-588 GAUUUGGAGAAUGAACCAGdTdT 1133 A-127027.1 AD-63377.1UCUCCAAAUCCCCGAGCACdTdT  800 A-127028.1  579-597 GUGCUCGGGGAUUUGGAGAdTdT1134 A-127029.1 AD-63383.1 CCGAGCACCGCCGGCUGAUdTdT  801 A-127030.1 590-608 AUCAGCCGGCGGUGCUCGGdTdT 1135 A-127031.1 AD-63342.1GGCUGAUGCUGAGCCCCGAdTdT  802 A-127032.1  602-620 UCGGGGCUCAGCAUCAGCCdTdT1136 A-127033.1 AD-63348.1 UGAGCCCCGAGGUGGUGCAdTdT  803 A-127034.1 611-629 UGCACCACCUCGGGGCUCAdTdT 1137 A-127035.1 AD-63354.1UGGUGCAGGCACUGCUGGUdTdT  804 A-127036.1  623-641 ACCAGCAGUGCCUGCACCAdTdT1138 A-127037.1 AD-63360.1 AGGCACUGCUGGUGGAGGAdTdT  805 A-127038.1 629-647 UCCUCCACCAGCAGUGCCUdTdT 1139 A-127039.1 AD-63366.1GUGGAGGAGCUGCUGUCCAdTdT  806 A-127040.1  640-658 UGGACAGCAGCUCCUCCACdTdT1140 A-127041.1 AD-63372.1 UGUCCACAGUCAACAGCUCdTdT  807 A-127042.1 653-671 GAGCUGUUGACUGUGGACAdTdT 1141 A-127043.1 AD-63378.1UCAACAGCUCGGCUGCCGUdTdT  808 A-127044.1  662-680 ACGGCAGCCGAGCUGUUGAdTdT1142 A-127045.1 AD-63384.1 UCGGCUGCCGUCCCCUACAdTdT  809 A-127046.1 670-688 UGUAGGGGACGGCAGCCGAdTdT 1143 A-127047.1 AD-63390.1AGUGGACCCCGAGGGCCUAdTdT  810 A-127048.1  702-720 UAGGCCCUCGGGGUCCACUdTdT1144 A-127049.1 AD-63396.1 AGGGCCUAGUGAUCCUGGAdTdT  811 A-127050.1 713-731 UCCAGGAUCACUAGGCCCUdTdT 1145 A-127051.1 AD-63402.1UAGUGAUCCUGGAAGCCAGdTdT  812 A-127052.1  719-737 CUGGCUUCCAGGAUCACUAdTdT1146 A-127053.1 AD-63408.1 AAGCCAGUGUGAAAGACAUdTdT  813 A-127054.1 731-749 AUGUCUUUCACACUGGCUUdTdT 1147 A-127055.1 AD-63414.1UGAAAGACAUAGCUGCAUUdTdT  814 A-127056.1  740-758 AAUGCAGCUAUGUCUUUCAdTdT1148 A-127057.1 AD-63420.1 UGCAUUGAAUUCCACGCUGdTdT  815 A-127058.1 753-771 CAGCGUGGAAUUCAAUGCAdTdT 1149 A-127059.1 AD-63426.1CUACAGCUACGUGGGCCAGdTdT  816 A-127060.1  783-801 CUGGCCCACGUAGCUGUAGdTdT1150 A-127061.1 AD-63385.1 CUACGUGGGCCAGGGCCAGdTdT  817 A-127062.1 789-807 CUGGCCCUGGCCCACGUAGdTdT 1151 A-127063.1 AD-63391.1AGGGCCAGGUCCUCCGGCUdTdT  818 A-127064.1  800-818 AGCCGGAGGACCUGGCCCUdTdT1152 A-127065.1 AD-63397.1 CCGGCUGAAGGGGCCUGACdTdT  819 A-127066.1 813-831 GUCAGGCCCCUUCAGCCGGdTdT 1153 A-127067.1 AD-63403.1GGGCCUGACCACCUGGCCUdTdT  820 A-127068.1  823-841 AGGCCAGGUGGUCAGGCCCdTdT1154 A-127069.1 AD-63409.1 CCACCUGGCCUCCAGCUGCdTdT  821 A-127070.1 831-849 GCAGCUGGAGGCCAGGUGGdTdT 1155 A-127071.1 AD-63415.1CCAGCUGCCUGUGGCACCUdTdT  822 A-127072.1  842-860 AGGUGCCACAGGCAGCUGGdTdT1156 A-127073.1 AD-63421.1 CUGUGGCACCUGCAGGGCCdTdT  823 A-127074.1 850-868 GGCCCUGCAGGUGCCACAGdTdT 1157 A-127075.1 AD-63427.1CUGCAGGGCCCCAAGGACCdTdT  824 A-127076.1  859-877 GGUCCUUGGGGCCCUGCAGdTdT1158 A-127077.1 AD-63386.1 CCAAGGACCUCAUGCUCAAdTdT  825 A-127078.1 869-887 UUGAGCAUGAGGUCCUUGGdTdT 1159 A-127079.1 AD-63392.1UGCUCAAACUCCGGCUGGAdTdT  826 A-127080.1  881-899 UCCAGCCGGAGUUUGAGCAdTdT1160 A-127081.1 AD-63398.1 CCGGCUGGAGUGGACGCUGdTdT  827 A-127082.1 891-909 CAGCGUCCACUCCAGCCGGdTdT 1161 A-127083.1 AD-63404.1GACGCUGGCAGAGUGCCGGdTdT  828 A-127084.1  903-921 CCGGCACUCUGCCAGCGUCdTdT1162 A-127085.1 AD-63410.1 GGCAGAGUGCCGGGACCGAdTdT  829 A-127086.1 909-927 UCGGUCCCGGCACUCUGCCdTdT 1163 A-127087.1 AD-63416.1ACCGACUGGCCAUGUAUGAdTdT  830 A-127088.1  923-941 UCAUACAUGGCCAGUCGGUdTdT1164 A-127089.1 AD-63422.1 CCAUGUAUGACGUGGCCGGdTdT  831 A-127090.1 932-950 CCGGCCACGUCAUACAUGGdTdT 1165 A-127091.1 AD-63428.1GUGGCCGGGCCCCUGGAGAdTdT  832 A-127092.1  943-961 UCUCCAGGGGCCCGGCCACdTdT1166 A-127093.1 AD-63387.1 CCCUGGAGAAGAGGCUCAUdTdT  833 A-127094.1 953-971 AUGAGCCUCUUCUCCAGGGdTdT 1167 A-127095.1 AD-63393.1AGAAGAGGCUCAUCACCUCdTdT  834 A-127096.1  959-977 GAGGUGAUGAGCCUCUUCUdTdT1168 A-127097.1 AD-63399.1 ACCUCGGUGUACGGCUGCAdTdT  835 A-127098.1 973-991 UGCAGCCGUACACCGAGGUdTdT 1169 A-127099.1 AD-63405.1ACGGCUGCAGCCGCCAGGAdTdT  836 A-127100.1  983-1001UCCUGGCGGCUGCAGCCGUdTdT 1170 A-127101.1 AD-63411.1GCCGCCAGGAGCCCGUGGUdTdT  837 A-127102.1  992-1010ACCACGGGCUCCUGGCGGCdTdT 1171 A-127103.1 AD-63417.1AGCCCGUGGUGGAGGUUCUdTdT  838 A-127104.1 1001-1019AGAACCUCCACCACGGGCUdTdT 1172 A-127105.1 AD-63423.1GUGGAGGUUCUGGCGUCGGdTdT  839 A-127106.1 1009-1027CCGACGCCAGAACCUCCACdTdT 1173 A-127107.1 AD-63429.1UGGCGUCGGGGGCCAUCAUdTdT  840 A-127108.1 1019-1037AUGAUGGCCCCCGACGCCAdTdT 1174 A-127109.1 AD-63388.1CCAUCAUGGCGGUCGUCUGdTdT  841 A-127110.1 1031-1049CAGACGACCGCCAUGAUGGdTdT 1175 A-127111.1 AD-63394.1GCGGUCGUCUGGAAGAAGGdTdT  842 A-127112.1 1039-1057CCUUCUUCCAGACGACCGCdTdT 1176 A-127113.1 AD-63400.1GGAAGAAGGGCCUGCACAGdTdT  843 A-127114.1 1049-1067CUGUGCAGGCCCUUCUUCCdTdT 1177 A-127115.1 AD-63406.1CCUGCACAGCUACUACGACdTdT  844 A-127116.1 1059-1077GUCGUAGUAGCUGUGCAGGdTdT 1178 A-127117.1 AD-63412.1ACUACGACCCCUUCGUGCUdTdT  845 A-127118.1 1070-1088AGCACGAAGGGGUCGUAGUdTdT 1179 A-127119.1 AD-63418.1CCUUCGUGCUCUCCGUGCAdTdT  846 A-127120.1 1079-1097UGCACGGAGAGCACGAAGGdTdT 1180 A-127121.1 AD-63424.1CCGUGCAGCCGGUGGUCUUdTdT  847 A-127122.1 1091-1109AAGACCACCGGCUGCACGGdTdT 1181 A-127123.1 AD-63430.1CGGUGGUCUUCCAGGCCUGdTdT  848 A-127124.1 1100-1118CAGGCCUGGAAGACCACCGdTdT 1182 A-127125.1 AD-63389.1AGGCCUGUGAAGUGAACCUdTdT  849 A-127126.1 1112-1130AGGUUCACUUCACAGGCCUdTdT 1183 A-127127.1 AD-63395.1AAGUGAACCUGACGCUGGAdTdT  850 A-127128.1 1121-1139UCCAGCGUCAGGUUCACUUdTdT 1184 A-127129.1 AD-63401.1GACGCUGGACAACAGGCUCdTdT  851 A-127130.1 1131-1149GAGCCUGUUGUCCAGCGUCdTdT 1185 A-127131.1 AD-63407.1ACAACAGGCUCGACUCCCAdTdT  852 A-127132.1 1139-1157UGGGAGUCGAGCCUGUUGUdTdT 1186 A-127133.1 AD-63413.1ACUCCCAGGGCGUCCUCAGdTdT  853 A-127134.1 1151-1169CUGAGGACGCCCUGGGAGUdTdT 1187 A-127135.1 AD-63419.1CCCCGUACUUCCCCAGCUAdTdT  854 A-127136.1 1172-1190UAGCUGGGGAAGUACGGGGdTdT 1188 A-127137.1 AD-63425.1UUCCCCAGCUACUACUCGCdTdT  855 A-127138.1 1180-1198GCGAGUAGUAGCUGGGGAAdTdT 1189 A-127139.1 AD-63431.1ACUACUCGCCCCAAACCCAdTdT  856 A-127140.1 1190-1208UGGGUUUGGGGCGAGUAGUdTdT 1190 A-127141.1 AD-63437.1CCCAAACCCACUGCUCCUGdTdT  857 A-127142.1 1199-1217CAGGAGCAGUGGGUUUGGGdTdT 1191 A-127143.1 AD-63443.1GCUCCUGGCACCUCACGGUdTdT  858 A-127144.1 1211-1229ACCGUGAGGUGCCAGGAGCdTdT 1192 A-127145.1 AD-63449.1ACCUCACGGUGCCCUCUCUdTdT  859 A-127146.1 1220-1238AGAGAGGGCACCGUGAGGUdTdT 1193 A-127147.1 AD-63455.1CUCUCUGGACUACGGCUUGdTdT  860 A-127148.1 1233-1251CAAGCCGUAGUCCAGAGAGdTdT 1194 A-127149.1 AD-63461.1GACUACGGCUUGGCCCUCUdTdT  861 A-127150.1 1240-1258AGAGGGCCAAGCCGUAGUCdTdT 1195 A-127151.1 AD-63467.1CCCUCUGGUUUGAUGCCUAdTdT  862 A-127152.1 1253-1271UAGGCAUCAAACCAGAGGGdTdT 1196 A-127153.1 AD-63473.1GUUUGAUGCCUAUGCACUGdTdT  863 A-127154.1 1260-1278CAGUGCAUAGGCAUCAAACdTdT 1197 A-127155.1 AD-63432.1GCACUGAGGAGGCAGAAGUdTdT  864 A-127156.1 1273-1291ACUUCUGCCUCCUCAGUGCdTdT 1198 A-127157.1 AD-63438.1GGAGGCAGAAGUAUGAUUUdTdT  865 A-127158.1 1280-1298AAAUCAUACUUCUGCCUCCdTdT 1199 A-127159.1 AD-63444.1AUGAUUUGCCGUGCACCCAdTdT  866 A-127160.1 1292-1310UGGGUGCACGGCAAAUCAUdTdT 1200 A-127161.1 AD-63450.1UGCACCCAGGGCCAGUGGAdTdT  867 A-127162.1 1303-1321UCCACUGGCCCUGGGUGCAdTdT 1201 A-127163.1 AD-63456.1GCCAGUGGACGAUCCAGAAdTdT  868 A-127164.1 1313-1331UUCUGGAUCGUCCACUGGCdTdT 1202 A-127165.1 AD-63462.1GGACGAUCCAGAACAGGAGdTdT  869 A-127166.1 1319-1337CUCCUGUUCUGGAUCGUCCdTdT 1203 A-127167.1 AD-63468.1ACAGGAGGCUGUGUGGCUUdTdT  870 A-127168.1 1331-1349AAGCCACACAGCCUCCUGUdTdT 1204 A-127169.1 AD-63474.1CUGUGUGGCUUGCGCAUCCdTdT  871 A-127170.1 1339-1357GGAUGCGCAAGCCACACAGdTdT 1205 A-127171.1 AD-63433.1UGCGCAUCCUGCAGCCCUAdTdT  872 A-127172.1 1349-1367UAGGGCUGCAGGAUGCGCAdTdT 1206 A-127173.1 AD-63439.1AGCCCUACGCCGAGAGGAUdTdT  873 A-127174.1 1361-1379AUCCUCUCGGCGUAGGGCUdTdT 1207 A-127175.1 AD-63445.1CCGAGAGGAUCCCCGUGGUdTdT  874 A-127176.1 1370-1388ACCACGGGGAUCCUCUCGGdTdT 1208 A-127177.1 AD-63451.1CCGUGGUGGCCACGGCCGGdTdT  875 A-127178.1 1382-1400CCGGCCGUGGCCACCACGGdTdT 1209 A-127179.1 AD-63457.1CCACGGCCGGGAUCACCAUdTdT  876 A-127180.1 1391-1409AUGGUGAUCCCGGCCGUGGdTdT 1210 A-127181.1 AD-63463.1GGAUCACCAUCAACUUCACdTdT  877 A-127182.1 1400-1418GUGAAGUUGAUGGUGAUCCdTdT 1211 A-127183.1 AD-63469.1UCAACUUCACCUCCCAGAUdTdT  878 A-127184.1 1409-1427AUCUGGGAGGUGAAGUUGAdTdT 1212 A-127185.1 AD-63475.1CCCAGAUCUCCCUCACCGGdTdT  879 A-127186.1 1421-1439CCGGUGAGGGAGAUCUGGGdTdT 1213 A-127187.1 AD-63434.1CCCUCACCGGGCCCGGUGUdTdT  880 A-127188.1 1430-1448ACACCGGGCCCGGUGAGGGdTdT 1214 A-127189.1 AD-63440.1CCCGGUGUGCGGGUGCACUdTdT  881 A-127190.1 1441-1459AGUGCACCCGCACACCGGGdTdT 1215 A-127191.1 AD-63446.1GCUUGUACAACCAGUCGGAdTdT  882 A-127192.1 1463-1481UCCGACUGGUUGUACAAGCdTdT 1216 A-127193.1 AD-63452.1ACAACCAGUCGGACCCCUGdTdT  883 A-127194.1 1469-1487CAGGGGUCCGACUGGUUGUdTdT 1217 A-127195.1 AD-63458.1ACCCCUGCCCUGGAGAGUUdTdT  884 A-127196.1 1481-1499AACUCUCCAGGGCAGGGGUdTdT 1218 A-127197.1 AD-63464.1CCUGGAGAGUUCCUCUGUUdTdT  885 A-127198.1 1489-1507AACAGAGGAACUCUCCAGGdTdT 1219 A-127199.1 AD-63470.1UCUGUUCUGUGAAUGGACUdTdT  886 A-127200.1 1502-1520AGUCCAUUCACAGAACAGAdTdT 1220 A-127201.1 AD-63476.1GAAUGGACUCUGUGUCCCUdTdT  887 A-127202.1 1512-1530AGGGACACAGAGUCCAUUCdTdT 1221 A-127203.1 AD-63435.1CUGUGUCCCUGCCUGUGAUdTdT  888 A-127204.1 1521-1539AUCACAGGCAGGGACACAGdTdT 1222 A-127205.1 AD-63441.1CUGCCUGUGAUGGGGUCAAdTdT  889 A-127206.1 1529-1547UUGACCCCAUCACAGGCAGdTdT 1223 A-127207.1 AD-63447.1GGUCAAGGACUGCCCCAACdTdT  890 A-127208.1 1542-1560GUUGGGGCAGUCCUUGACCdTdT 1224 A-127209.1 AD-63453.1UGCCCCAACGGCCUGGAUGdTdT  891 A-127210.1 1552-1570CAUCCAGGCCGUUGGGGCAdTdT 1225 A-127211.1 AD-63459.1CGGCCUGGAUGAGAGAAACdTdT  892 A-127212.1 1560-1578GUUUCUCUCAUCCAGGCCGdTdT 1226 A-127213.1 AD-63465.1GAGAGAAACUGCGUUUGCAdTdT  893 A-127214.1 1570-1588UGCAAACGCAGUUUCUCUCdTdT 1227 A-127215.1 AD-63471.1UUUGCAGAGCCACAUUCCAdTdT  894 A-127216.1 1583-1601UGGAAUGUGGCUCUGCAAAdTdT 1228 A-127217.1 AD-63477.1GCCACAUUCCAGUGCAAAGdTdT  895 A-127218.1 1591-1609CUUUGCACUGGAAUGUGGCdTdT 1229 A-127219.1 AD-63436.1GUGCAAAGAGGACAGCACAdTdT  896 A-127220.1 1602-1620UGUGCUGUCCUCUUUGCACdTdT 1230 A-127221.1 AD-63442.1GAGGACAGCACAUGCAUCUdTdT  897 A-127222.1 1609-1627AGAUGCAUGUGCUGUCCUCdTdT 1231 A-127223.1 AD-63448.1GCAUCUCACUGCCCAAGGUdTdT  898 A-127224.1 1622-1640ACCUUGGGCAGUGAGAUGCdTdT 1232 A-127225.1 AD-63454.1GCCCAAGGUCUGUGAUGGGdTdT  899 A-127226.1 1632-1650CCCAUCACAGACCUUGGGCdTdT 1233 A-127227.1 AD-63460.1UGUGAUGGGCAGCCUGAUUdTdT  900 A-127228.1 1642-1660AAUCAGGCUGCCCAUCACAdTdT 1234 A-127229.1 AD-63466.1GCAGCCUGAUUGUCUCAACdTdT  901 A-127230.1 1650-1668GUUGAGACAAUCAGGCUGCdTdT 1235 A-127231.1 AD-63472.1GUCUCAACGGCAGCGACGAdTdT  902 A-127232.1 1661-1679UCGUCGCUGCCGUUGAGACdTdT 1236 A-127233.1 AD-63478.1GCGACGAAGAGCAGUGCCAdTdT  903 A-127234.1 1673-1691UGGCACUGCUCUUCGUCGCdTdT 1237 A-127235.1 AD-63484.1AGCAGUGCCAGGAAGGGGUdTdT  904 A-127236.1 1682-1700ACCCCUUCCUGGCACUGCUdTdT 1238 A-127237.1 AD-63490.1GAAGGGGUGCCAUGUGGGAdTdT  905 A-127238.1 1693-1711UCCCACAUGGCACCCCUUCdTdT 1239 A-127239.1 AD-63496.1CCAUGUGGGACAUUCACCUdTdT  906 A-127240.1 1702-1720AGGUGAAUGUCCCACAUGGdTdT 1240 A-127241.1 AD-63502.1CAUUCACCUUCCAGUGUGAdTdT  907 A-127242.1 1712-1730UCACACUGGAAGGUGAAUGdTdT 1241 A-127243.1 AD-63508.1CAGUGUGAGGACCGGAGCUdTdT  908 A-127244.1 1723-1741AGCUCCGGUCCUCACACUGdTdT 1242 A-127245.1 AD-63514.1GACCGGAGCUGCGUGAAGAdTdT  909 A-127246.1 1732-1750UCUUCACGCAGCUCCGGUCdTdT 1243 A-127247.1 AD-63520.1CUGCGUGAAGAAGCCCAACdTdT  910 A-127248.1 1740-1758GUUGGGCUUCUUCACGCAGdTdT 1244 A-127249.1 AD-63479.1AGCCCAACCCGCAGUGUGAdTdT  911 A-127250.1 1751-1769UCACACUGCGGGUUGGGCUdTdT 1245 A-127251.1 AD-63485.1CAGUGUGAUGGGCGGCCCGdTdT  912 A-127252.1 1762-1780CGGGCCGCCCAUCACACUGdTdT 1246 A-127253.1 AD-63491.1GCGGCCCGACUGCAGGGACdTdT  913 A-127254.1 1773-1791GUCCCUGCAGUCGGGCCGCdTdT 1247 A-127255.1 AD-63497.1CUGCAGGGACGGCUCGGAUdTdT  914 A-127256.1 1782-1800AUCCGAGCCGUCCCUGCAGdTdT 1248 A-127257.1 AD-63503.1ACGGCUCGGAUGAGGAGCAdTdT  915 A-127258.1 1790-1808UGCUCCUCAUCCGAGCCGUdTdT 1249 A-127259.1 AD-63509.1UGAGGAGCACUGUGACUGUdTdT  916 A-127260.1 1800-1818ACAGUCACAGUGCUCCUCAdTdT 1250 A-127261.1 AD-63515.1CUGUGACUGUGGCCUCCAGdTdT  917 A-127262.1 1809-1827CUGGAGGCCACAGUCACAGdTdT 1251 A-127263.1 AD-63521.1GCCUCCAGGGCCCCUCCAGdTdT  918 A-127264.1 1820-1838CUGGAGGGGCCCUGGAGGCdTdT 1252 A-127265.1 AD-63480.1CCCCUCCAGCCGCAUUGUUdTdT  919 A-127266.1 1830-1848AACAAUGCGGCUGGAGGGGdTdT 1253 A-127267.1 AD-63486.1CCGCAUUGUUGGUGGAGCUdTdT  920 A-127268.1 1839-1857AGCUCCACCAACAAUGCGGdTdT 1254 A-127269.1 AD-63492.1GUGGAGCUGUGUCCUCCGAdTdT  921 A-127270.1 1850-1868UCGGAGGACACAGCUCCACdTdT 1255 A-127271.1 AD-63498.1CUCCGAGGGUGAGUGGCCAdTdT  922 A-127272.1 1863-1881UGGCCACUCACCCUCGGAGdTdT 1256 A-127273.1 AD-63504.1GGGUGAGUGGCCAUGGCAGdTdT  923 A-127274.1 1869-1887CUGCCAUGGCCACUCACCCdTdT 1257 A-127275.1 AD-63510.1AUGGCAGGCCAGCCUCCAGdTdT  924 A-127276.1 1881-1899CUGGAGGCUGGCCUGCCAUdTdT 1258 A-127277.1 AD-63516.1CCUCCAGGUUCGGGGUCGAdTdT  925 A-127278.1 1893-1911UCGACCCCGAACCUGGAGGdTdT 1259 A-127279.1 AD-63522.1GGUUCGGGGUCGACACAUCdTdT  926 A-127280.1 1899-1917GAUGUGUCGACCCCGAACCdTdT 1260 A-127281.1 AD-63481.1ACAUCUGUGGGGGGGCCCUdTdT  927 A-127282.1 1913-1931AGGGCCCCCCCACAGAUGUdTdT 1261 A-127283.1 AD-63487.1GUGGGGGGGCCCUCAUCGCdTdT  928 A-127284.1 1919-1937GCGAUGAGGGCCCCCCCACdTdT 1262 A-127285.1 AD-63493.1AUCGCUGACCGCUGGGUGAdTdT  929 A-127286.1 1933-1951UCACCCAGCGGUCAGCGAUdTdT 1263 A-127287.1 AD-63499.1ACCGCUGGGUGAUAACAGCdTdT  930 A-127288.1 1940-1958GCUGUUAUCACCCAGCGGUdTdT 1264 A-127289.1 AD-63505.1UGAUAACAGCUGCCCACUGdTdT  931 A-127290.1 1949-1967CAGUGGGCAGCUGUUAUCAdTdT 1265 A-127291.1 AD-63511.1CCCACUGCUUCCAGGAGGAdTdT  932 A-127292.1 1961-1979UCCUCCUGGAAGCAGUGGGdTdT 1266 A-127293.1 AD-63517.1CCAGGAGGACAGCAUGGCCdTdT  933 A-127294.1 1971-1989GGCCAUGCUGUCCUCCUGGdTdT 1267 A-127295.1 AD-63523.1ACAGCAUGGCCUCCACGGUdTdT  934 A-127296.1 1979-1997ACCGUGGAGGCCAUGCUGUdTdT 1268 A-127297.1 AD-63482.1CCACGGUGCUGUGGACCGUdTdT  935 A-127298.1 1991-2009ACGGUCCACAGCACCGUGGdTdT 1269 A-127299.1 AD-63488.1GGACCGUGUUCCUGGGCAAdTdT  936 A-127300.1 2003-2021UUGCCCAGGAACACGGUCCdTdT 1270 A-127301.1 AD-63494.1UCCUGGGCAAGGUGUGGCAdTdT  937 A-127302.1 2012-2030UGCCACACCUUGCCCAGGAdTdT 1271 A-127303.1 AD-63500.1GUGUGGCAGAACUCGCGCUdTdT  938 A-127304.1 2023-2041AGCGCGAGUUCUGCCACACdTdT 1272 A-127305.1 AD-63506.1GAACUCGCGCUGGCCUGGAdTdT  939 A-127306.1 2031-2049UCCAGGCCAGCGCGAGUUCdTdT 1273 A-127307.1 AD-63512.1GGCCUGGAGAGGUGUCCUUdTdT  940 A-127308.1 2042-2060AAGGACACCUCUCCAGGCCdTdT 1274 A-127309.1 AD-63518.1AGGUGUCCUUCAAGGUGAGdTdT  941 A-127310.1 2051-2069CUCACCUUGAAGGACACCUdTdT 1275 A-127311.1 AD-63524.1CAAGGUGAGCCGCCUGCUCdTdT  942 A-127312.1 2061-2079GAGCAGGCGGCUCACCUUGdTdT 1276 A-127313.1 AD-63483.1GCCUGCUCCUGCACCCGUAdTdT  943 A-127314.1 2072-2090UACGGGUGCAGGAGCAGGCdTdT 1277 A-127315.1 AD-63489.1GCACCCGUACCACGAAGAGdTdT  944 A-127316.1 2082-2100CUCUUCGUGGUACGGGUGCdTdT 1278 A-127317.1 AD-63495.1CCACGAAGAGGACAGCCAUdTdT  945 A-127318.1 2091-2109AUGGCUGUCCUCUUCGUGGdTdT 1279 A-127319.1 AD-63501.1AGGACAGCCAUGACUACGAdTdT  946 A-127320.1 2099-2117UCGUAGUCAUGGCUGUCCUdTdT 1280 A-127321.1 AD-63507.1ACUACGACGUGGCGCUGCUdTdT  947 A-127322.1 2111-2129AGCAGCGCCACGUCGUAGUdTdT 1281 A-127323.1 AD-63513.1UGGCGCUGCUGCAGCUCGAdTdT  948 A-127324.1 2120-2138UCGAGCUGCAGCAGCGCCAdTdT 1282 A-127325.1 AD-63519.1AGCUCGACCACCCGGUGGUdTdT  949 A-127326.1 2132-2150ACCACCGGGUGGUCGAGCUdTdT 1283 A-127327.1 AD-63525.1CCGGUGGUGCGCUCGGCCGdTdT  950 A-127328.1 2143-2161CGGCCGAGCGCACCACCGGdTdT 1284 A-127329.1 AD-63531.1UGCGCUCGGCCGCCGUGCGdTdT  951 A-127330.1 2150-2168CGCACGGCGGCCGAGCGCAdTdT 1285 A-127331.1 AD-63537.1CCGUGCGCCCCGUCUGCCUdTdT  952 A-127332.1 2162-2180AGGCAGACGGGGCGCACGGdTdT 1286 A-127333.1 AD-63543.1CCGUCUGCCUGCCCGCGCGdTdT  953 A-127334.1 2171-2189CGCGCGGGCAGGCAGACGGdTdT 1287 A-127335.1 AD-63549.1CCGCGCGCUCCCACUUCUUdTdT  954 A-127336.1 2183-2201AAGAAGUGGGAGCGCGCGGdTdT 1288 A-127337.1 AD-63555.1CCCACUUCUUCGAGCCCGGdTdT  955 A-127338.1 2192-2210CCGGGCUCGAAGAAGUGGGdTdT 1289 A-127339.1 AD-63561.1GAGCCCGGCCUGCACUGCUdTdT  956 A-127340.1 2203-2221AGCAGUGCAGGCCGGGCUCdTdT 1290 A-127341.1 AD-63567.1GGCCUGCACUGCUGGAUUAdTdT  957 A-127342.1 2209-2227UAAUCCAGCAGUGCAGGCCdTdT 1291 A-127343.1 AD-63526.1UGGAUUACGGGCUGGGGCGdTdT  958 A-127344.1 2221-2239CGCCCCAGCCCGUAAUCCAdTdT 1292 A-127345.1 AD-63532.1GCUGGGGCGCCUUGCGCGAdTdT  959 A-127346.1 2231-2249UCGCGCAAGGCGCCCCAGCdTdT 1293 A-127347.1 AD-63538.1UGCGCGAGGGCGGCCCCAUdTdT  960 A-127348.1 2243-2261AUGGGGCCGCCCUCGCGCAdTdT 1294 A-127349.1 AD-63544.1AGGGCGGCCCCAUCAGCAAdTdT  961 A-127350.1 2249-2267UUGCUGAUGGGGCCGCCCUdTdT 1295 A-127351.1 AD-63550.1UCAGCAACGCUCUGCAGAAdTdT  962 A-127352.1 2261-2279UUCUGCAGAGCGUUGCUGAdTdT 1296 A-127353.1 AD-63556.1UGCAGAAAGUGGAUGUGCAdTdT  963 A-127354.1 2273-2291UGCACAUCCACUUUCUGCAdTdT 1297 A-127355.1 AD-63562.1AAGUGGAUGUGCAGUUGAUdTdT  964 A-127356.1 2279-2297AUCAACUGCACAUCCACUUdTdT 1298 A-127357.1 AD-63568.1GCAGUUGAUCCCACAGGACdTdT  965 A-127358.1 2289-2307GUCCUGUGGGAUCAACUGCdTdT 1299 A-127359.1 AD-63527.1CACAGGACCUGUGCAGCGAdTdT  966 A-127360.1 2300-2318UCGCUGCACAGGUCCUGUGdTdT 1300 A-127361.1 AD-63533.1GCAGCGAGGUCUAUCGCUAdTdT  967 A-127362.1 2312-2330UAGCGAUAGACCUCGCUGCdTdT 1301 A-127363.1 AD-63539.1GUCUAUCGCUACCAGGUGAdTdT  968 A-127364.1 2320-2338UCACCUGGUAGCGAUAGACdTdT 1302 A-127365.1 AD-63545.1CCAGGUGACGCCACGCAUGdTdT  969 A-127366.1 2331-2349CAUGCGUGGCGUCACCUGGdTdT 1303 A-127367.1 AD-63551.1CCACGCAUGCUGUGUGCCGdTdT  970 A-127368.1 2341-2359CGGCACACAGCAUGCGUGGdTdT 1304 A-127369.1 AD-63557.1CUGUGUGCCGGCUACCGCAdTdT  971 A-127370.1 2350-2368UGCGGUAGCCGGCACACAGdTdT 1305 A-127371.1 AD-63563.1ACCGCAAGGGCAAGAAGGAdTdT  972 A-127372.1 2363-2381UCCUUCUUGCCCUUGCGGUdTdT 1306 A-127373.1 AD-63569.1GCAAGAAGGAUGCCUGUCAdTdT  973 A-127374.1 2372-2390UGACAGGCAUCCUUCUUGCdTdT 1307 A-127375.1 AD-63528.1GCCUGUCAGGGUGACUCAGdTdT  974 A-127376.1 2383-2401CUGAGUCACCCUGACAGGCdTdT 1308 A-127377.1 AD-63534.1GUGACUCAGGUGGUCCGCUdTdT  975 A-127378.1 2393-2411AGCGGACCACCUGAGUCACdTdT 1309 A-127379.1 AD-63540.1GUGGUCCGCUGGUGUGCAAdTdT  976 A-127380.1 2402-2420UUGCACACCAGCGGACCACdTdT 1310 A-127381.1 AD-63546.1UGGUGUGCAAGGCACUCAGdTdT  977 A-127382.1 2411-2429CUGAGUGCCUUGCACACCAdTdT 1311 A-127383.1 AD-63552.1GCACUCAGUGGCCGCUGGUdTdT  978 A-127384.1 2422-2440ACCAGCGGCCACUGAGUGCdTdT 1312 A-127385.1 AD-63558.1GCCGCUGGUUCCUGGCGGGdTdT  979 A-127386.1 2432-2450CCCGCCAGGAACCAGCGGCdTdT 1313 A-127387.1 AD-63564.1UCCUGGCGGGGCUGGUCAGdTdT  980 A-127388.1 2441-2459CUGACCAGCCCCGCCAGGAdTdT 1314 A-127389.1 AD-63570.1GCUGGUCAGCUGGGGCCUGdTdT  981 A-127390.1 2451-2469CAGGCCCCAGCUGACCAGCdTdT 1315 A-127391.1 AD-63529.1GGGCCUGGGCUGUGGCCGGdTdT  982 A-127392.1 2463-2481CCGGCCACAGCCCAGGCCCdTdT 1316 A-127393.1 AD-63535.1GGCUGUGGCCGGCCUAACUdTdT  983 A-127394.1 2470-2488AGUUAGGCCGGCCACAGCCdTdT 1317 A-127395.1 AD-63541.1CUAACUACUUCGGCGUCUAdTdT  984 A-127396.1 2483-2501UAGACGCCGAAGUAGUUAGdTdT 1318 A-127397.1 AD-63547.1CGGCGUCUACACCCGCAUCdTdT  985 A-127398.1 2493-2511GAUGCGGGUGUAGACGCCGdTdT 1319 A-127399.1 AD-63553.1ACACCCGCAUCACAGGUGUdTdT  986 A-127400.1 2501-2519ACACCUGUGAUGCGGGUGUdTdT 1320 A-127401.1 AD-63559.1ACAGGUGUGAUCAGCUGGAdTdT  987 A-127402.1 2512-2530UCCAGCUGAUCACACCUGUdTdT 1321 A-127403.1 AD-63565.1UCAGCUGGAUCCAGCAAGUdTdT  988 A-127404.1 2522-2540ACUUGCUGGAUCCAGCUGAdTdT 1322 A-127405.1 AD-63571.1CAGCAAGUGGUGACCUGAGdTdT  989 A-127406.1 2533-2551CUCAGGUCACCACUUGCUGdTdT 1323 A-127407.1 AD-63530.1UGACCUGAGGAACUGCCCCdTdT  990 A-127408.1 2543-2561GGGGCAGUUCCUCAGGUCAdTdT 1324 A-127409.1 AD-63536.1GGAACUGCCCCCCUGCAAAdTdT  991 A-127410.1 2551-2569UUUGCAGGGGGGCAGUUCCdTdT 1325 A-127411.1 AD-63542.1CUGCAAAGCAGGGCCCACCdTdT  992 A-127412.1 2563-2581GGUGGGCCCUGCUUUGCAGdTdT 1326 A-127413.1 AD-63548.1GCAGGGCCCACCUCCUGGAdTdT  993 A-127414.1 2570-2588UCCAGGAGGUGGGCCCUGCdTdT 1327 A-127415.1 AD-63554.1CCUCCUGGACUCAGAGAGCdTdT  994 A-127416.1 2580-2598GCUCUCUGAGUCCAGGAGGdTdT 1328 A-127417.1 AD-63560.1CUCAGAGAGCCCAGGGCAAdTdT  995 A-127418.1 2589-2607UUGCCCUGGGCUCUCUGAGdTdT 1329 A-127419.1 AD-63566.1CCAGGGCAACUGCCAAGCAdTdT  996 A-127420.1 2599-2617UGCUUGGCAGUUGCCCUGGdTdT 1330 A-127421.1 AD-63572.1GGACAAGUAUUCUGGCGGGdTdT  997 A-127422.1 2621-2639CCCGCCAGAAUACUUGUCCdTdT 1331 A-127423.1 AD-63578.1CUGGCGGGGGGUGGGGGAGdTdT  998 A-127424.1 2632-2650CUCCCCCACCCCCCGCCAGdTdT 1332 A-127425.1 AD-63584.1GGGUGGGGGAGAGAGCAGGdTdT  999 A-127426.1 2640-2658CCUGCUCUCUCCCCCACCCdTdT 1333 A-127427.1 AD-63590.1AGAGAGCAGGCCCUGUGGUdTdT 1000 A-127428.1 2649-2667ACCACAGGGCCUGCUCUCUdTdT 1334 A-127429.1 AD-63596.1CCCUGUGGUGGCAGGAGGUdTdT 1001 A-127430.1 2659-2677ACCUCCUGCCACCACAGGGdTdT 1335 A-127431.1 AD-63602.1GGAGGUGGCAUCUUGUCUCdTdT 1002 A-127432.1 2672-2690GAGACAAGAUGCCACCUCCdTdT 1336 A-127433.1 AD-63608.1CAUCUUGUCUCGUCCCUGAdTdT 1003 A-127434.1 2680-2698UCAGGGACGAGACAAGAUGdTdT 1337 A-127435.1 AD-63614.1CCCUGAUGUCUGCUCCAGUdTdT 1004 A-127436.1 2693-2711ACUGGAGCAGACAUCAGGGdTdT 1338 A-127437.1 AD-63573.1CUGCUCCAGUGAUGGCAGGdTdT 1005 A-127438.1 2702-2720CCUGCCAUCACUGGAGCAGdTdT 1339 A-127439.1 AD-63579.1AUGGCAGGAGGAUGGAGAAdTdT 1006 A-127440.1 2713-2731UUCUCCAUCCUCCUGCCAUdTdT 1340 A-127441.1 AD-63585.1GGAUGGAGAAGUGCCAGCAdTdT 1007 A-127442.1 2722-2740UGCUGGCACUUCUCCAUCCdTdT 1341 A-127443.1 AD-63591.1UGCCAGCAGCUGGGGGUCAdTdT 1008 A-127444.1 2733-2751UGACCCCCAGCUGCUGGCAdTdT 1342 A-127445.1 AD-63597.1AGCUGGGGGUCAAGACGUCdTdT 1009 A-127446.1 2740-2758GACGUCUUGACCCCCAGCUdTdT 1343 A-127447.1 AD-63603.1UCAAGACGUCCCCUGAGGAdTdT 1010 A-127448.1 2749-2767UCCUCAGGGGACGUCUUGAdTdT 1344 A-127449.1 AD-63609.1CCCUGAGGACCCAGGCCCAdTdT 1011 A-127450.1 2759-2777UGGGCCUGGGUCCUCAGGGdTdT 1345 A-127451.1 AD-63615.1GCCCACACCCAGCCCUUCUdTdT 1012 A-127452.1 2773-2791AGAAGGGCUGGGUGUGGGCdTdT 1346 A-127453.1 AD-63574.1AGCCCUUCUGCCUCCCAAUdTdT 1013 A-127454.1 2783-2801AUUGGGAGGCAGAAGGGCUdTdT 1347 A-127455.1 AD-63580.1CCUCCCAAUUCUCUCUCCUdTdT 1014 A-127456.1 2793-2811AGGAGAGAGAAUUGGGAGGdTdT 1348 A-127457.1 AD-63586.1CUCUCUCCUCCGUCCCCUUdTdT 1015 A-127458.1 2803-2821AAGGGGACGGAGGAGAGAGdTdT 1349 A-127459.1 AD-63592.1UCCGUCCCCUUCCUCCACUdTdT 1016 A-127460.1 2811-2829AGUGGAGGAAGGGGACGGAdTdT 1350 A-127461.1 AD-63598.1CUUCCUCCACUGCUGCCUAdTdT 1017 A-127462.1 2819-2837UAGGCAGCAGUGGAGGAAGdTdT 1351 A-127463.1 AD-63604.1CUGCCUAAUGCAAGGCAGUdTdT 1018 A-127464.1 2831-2849ACUGCCUUGCAUUAGGCAGdTdT 1352 A-127465.1 AD-63610.1GCAAGGCAGUGGCUCAGCAdTdT 1019 A-127466.1 2840-2858UGCUGAGCCACUGCCUUGCdTdT 1353 A-127467.1 AD-63616.1UGGCUCAGCAGCAAGAAUGdTdT 1020 A-127468.1 2849-2867CAUUCUUGCUGCUGAGCCAdTdT 1354 A-127469.1 AD-63575.1CAAGAAUGCUGGUUCUACAdTdT 1021 A-127470.1 2860-2878UGUAGAACCAGCAUUCUUGdTdT 1355 A-127471.1 AD-63581.1UGGUUCUACAUCCCGAGGAdTdT 1022 A-127472.1 2869-2887UCCUCGGGAUGUAGAACCAdTdT 1356 A-127473.1 AD-63587.1CCCGAGGAGUGUCUGAGGUdTdT 1023 A-127474.1 2880-2898ACCUCAGACACUCCUCGGGdTdT 1357 A-127475.1 AD-63593.1GUCUGAGGUGCGCCCCACUdTdT 1024 A-127476.1 2890-2908AGUGGGGCGCACCUCAGACdTdT 1358 A-127477.1 AD-63599.1GCCCCACUCUGUACAGAGGdTdT 1025 A-127478.1 2901-2919CCUCUGUACAGAGUGGGGCdTdT 1359 A-127479.1 AD-63605.1CUGUACAGAGGCUGUUUGGdTdT 1026 A-127480.1 2909-2927CCAAACAGCCUCUGUACAGdTdT 1360 A-127481.1 AD-63611.1CUGUUUGGGCAGCCUUGCCdTdT 1027 A-127482.1 2920-2938GGCAAGGCUGCCCAAACAGdTdT 1361 A-127483.1 AD-63617.1CUUGCCUCCAGAGAGCAGAdTdT 1028 A-127484.1 2933-2951UCUGCUCUCUGGAGGCAAGdTdT 1362 A-127485.1 AD-63576.1UCCAGAGAGCAGAUUCCAGdTdT 1029 A-127486.1 2939-2957CUGGAAUCUGCUCUCUGGAdTdT 1363 A-127487.1 AD-63582.1GAUUCCAGCUUCGGAAGCCdTdT 1030 A-127488.1 2950-2968GGCUUCCGAAGCUGGAAUCdTdT 1364 A-127489.1 AD-63588.1GAAUGGAAGGUGCUCCCAUdTdT 1031 A-127490.1 2991-3009AUGGGAGCACCUUCCAUUCdTdT 1365 A-127491.1 AD-63594.1GUGCUCCCAUCGGAGGGGAdTdT 1032 A-127492.1 3000-3018UCCCCUCCGAUGGGAGCACdTdT 1366 A-127493.1 AD-63600.1UCGGAGGGGACCCUCAGAGdTdT 1033 A-127494.1 3009-3027CUCUGAGGGUCCCCUCCGAdTdT 1367 A-127495.1 AD-63606.1CCCUCAGAGCCCUGGAGACdTdT 1034 A-127496.1 3019-3037GUCUCCAGGGCUCUGAGGGdTdT 1368 A-127497.1 AD-63612.1GAGACUGCCAGGUGGGCCUdTdT 1035 A-127498.1 3033-3051AGGCCCACCUGGCAGUCUCdTdT 1369 A-127499.1 AD-63618.1AGGUGGGCCUGCUGCCACUdTdT 1036 A-127500.1 3042-3060AGUGGCAGCAGGCCCACCUdTdT 1370 A-127501.1 AD-63577.1CUGCCACUGUAAGCCAAAAdTdT 1037 A-127502.1 3053-3071UUUUGGCUUACAGUGGCAGdTdT 1371 A-127503.1 AD-63583.1CUGUAAGCCAAAAGGUGGGdTdT 1038 A-127504.1 3059-3077CCCACCUUUUGGCUUACAGdTdT 1372 A-127505.1 AD-63589.1GUGGGGAAGUCCUGACUCCdTdT 1039 A-127506.1 3073-3091GGAGUCAGGACUUCCCCACdTdT 1373 A-127507.1 AD-63595.1CCUGACUCCAGGGUCCUUGdTdT 1040 A-127508.1 3083-3101CAAGGACCCUGGAGUCAGGdTdT 1374 A-127509.1 AD-63601.1GGGUCCUUGCCCCACCCCUdTdT 1041 A-127510.1 3093-3111AGGGGUGGGGCAAGGACCCdTdT 1375 A-127511.1 AD-63607.1GCCCCACCCCUGCCUGCCAdTdT 1042 A-127512.1 3101-3119UGGCAGGCAGGGGUGGGGCdTdT 1376 A-127513.1 AD-63613.1CCUGCCACCUGGGCCCUCAdTdT 1043 A-127514.1 3113-3131UGAGGGCCCAGGUGGCAGGdTdT 1377 A-127515.1 AD-63619.1CUGGGCCCUCACAGCCCAGdTdT 1044 A-127516.1 3121-3139CUGGGCUGUGAGGGCCCAGdTdT 1378 A-127517.1 AD-63620.1UCACAGCCCAGACCCUCACdTdT 1045 A-127518.1 3129-3147GUGAGGGUCUGGGCUGUGAdTdT 1379 A-127519.1 AD-63621.1CUCACUGGGAGGUGAGCUCdTdT 1046 A-127520.1 3143-3161GAGCUCACCUCCCAGUGAGdTdT 1380 A-127521.1 AD-63622.1GGUGAGCUCAGCUGCCCUUdTdT 1047 A-127522.1 3153-3171AAGGGCAGCUGAGCUCACCdTdT 1381 A-127523.1 AD-63623.1UGGAAUAAAGCUGCCUGAUdTdT 1048 A-127524.1 3172-3190AUCAGGCAGCUUUAUUCCAdTdT 1382 A-127525.1

TABLE 13 TMPRSS6 single dose screen (10 nM) in Hep3B cells with dTmodified siRNAs Avg % message Duplex ID remaining SD AD-63290.1 122.818.0 AD-63296.1 87.4 6.0 AD-63302.1 71.4 16.9 AD-63308.1 82.1 10.3AD-63314.1 59.1 5.3 AD-63320.1 90.7 4.5 AD-63326.1 121.0 18.2 AD-63332.1114.4 11.6 AD-63291.1 84.7 15.0 AD-63297.1 82.8 3.9 AD-63303.1 67.6 5.5AD-63309.1 55.8 6.5 AD-63315.1 64.2 7.4 AD-63321.1 85.8 6.4 AD-63327.191.9 14.9 AD-63333.1 76.4 5.2 AD-63292.1 54.4 22.9 AD-63298.1 54.6 5.0AD-63304.1 24.6 7.3 AD-63310.1 23.3 0.6 AD-63316.1 50.9 7.2 AD-63322.153.7 10.5 AD-63328.1 29.2 2.3 AD-63334.1 28.5 1.2 AD-63293.1 50.9 6.8AD-63299.1 85.5 2.3 AD-63305.1 43.0 7.2 AD-63311.1 28.9 2.6 AD-63317.140.9 2.7 AD-63323.1 40.2 7.3 AD-63329.1 27.9 12.0 AD-63335.1 82.0 4.2AD-63294.1 21.8 1.0 AD-63300.1 32.3 8.0 AD-63306.1 32.9 8.3 AD-63312.126.5 4.6 AD-63318.1 31.3 2.4 AD-63324.1 25.7 1.9 AD-63330.1 24.5 2.0AD-63336.1 36.1 8.6 AD-63295.1 29.2 1.8 AD-63301.1 28.9 5.2 AD-63307.168.8 10.6 AD-63313.1 90.2 8.2 AD-63319.1 21.9 3.3 AD-63325.1 26.1 4.8AD-63331.1 36.7 4.5 AD-63337.1 67.7 9.3 AD-63343.1 83.9 15.0 AD-63349.171.6 3.5 AD-63355.1 62.8 10.4 AD-63361.1 56.0 3.3 AD-63367.1 49.3 8.7AD-63373.1 54.1 8.2 AD-63379.1 47.5 6.3 AD-63338.1 28.0 2.8 AD-63344.129.7 5.7 AD-63350.1 23.0 2.3 AD-63356.1 81.5 13.7 AD-63362.1 19.7 2.9AD-63368.1 42.2 4.7 AD-63374.1 24.5 2.0 AD-63380.1 24.9 4.9 AD-63339.128.9 10.1 AD-63345.1 29.9 5.6 AD-63351.1 20.4 3.7 AD-63357.1 35.8 6.8AD-63363.1 30.4 2.5 AD-63369.1 29.0 3.1 AD-63375.1 36.6 2.4 AD-63381.129.1 4.3 AD-63340.1 40.4 18.8 AD-63346.1 36.4 3.5 AD-63352.1 25.8 3.9AD-63358.1 42.6 8.1 AD-63364.1 48.1 6.6 AD-63370.1 24.6 2.8 AD-63376.122.1 4.2 AD-63382.1 31.0 7.5 AD-63341.1 37.6 13.7 AD-63347.1 27.6 2.0AD-63353.1 76.4 14.5 AD-63359.1 25.3 1.1 AD-63365.1 27.3 3.4 AD-63371.116.3 1.3 AD-63377.1 65.4 7.1 AD-63383.1 72.2 7.0 AD-63342.1 30.8 7.3AD-63348.1 72.7 9.2 AD-63354.1 38.7 5.0 AD-63360.1 28.7 3.0 AD-63366.130.9 6.8 AD-63372.1 84.0 9.0 AD-63378.1 64.1 8.6 AD-63384.1 38.0 2.6AD-63390.1 48.3 10.6 AD-63396.1 45.6 7.0 AD-63402.1 42.0 9.9 AD-63408.140.4 9.1 AD-63414.1 23.8 6.2 AD-63420.1 55.3 5.2 AD-63426.1 61.6 8.5AD-63385.1 61.6 10.2 AD-63391.1 38.0 3.1 AD-63397.1 66.7 16.8 AD-63403.177.2 15.4 AD-63409.1 60.3 10.7 AD-63415.1 35.0 5.4 AD-63421.1 60.6 2.9AD-63427.1 40.5 7.2 AD-63386.1 42.0 7.4 AD-63392.1 34.2 3.1 AD-63398.162.6 18.5 AD-63404.1 65.9 8.1 AD-63410.1 19.7 4.0 AD-63416.1 51.3 9.0AD-63422.1 59.3 2.7 AD-63428.1 58.2 9.7 AD-63387.1 42.2 4.8 AD-63393.127.9 4.4 AD-63399.1 49.6 8.4 AD-63405.1 72.5 9.3 AD-63411.1 45.4 14.9AD-63417.1 36.7 9.4 AD-63423.1 76.8 4.9 AD-63429.1 77.8 14.4 AD-63388.137.4 4.4 AD-63394.1 31.5 4.6 AD-63400.1 60.9 28.6 AD-63406.1 40.7 14.3AD-63412.1 22.0 7.0 AD-63418.1 22.8 4.3 AD-63424.1 25.5 2.8 AD-63430.121.5 3.2 AD-63389.1 34.4 5.3 AD-63395.1 31.1 0.7 AD-63401.1 44.3 9.5AD-63407.1 41.5 4.9 AD-63413.1 52.4 6.4 AD-63419.1 26.3 5.6 AD-63425.178.8 4.6 AD-63431.1 32.8 6.6 AD-63437.1 42.3 1.4 AD-63443.1 56.4 8.9AD-63449.1 26.0 5.9 AD-63455.1 28.0 9.7 AD-63461.1 32.1 11.1 AD-63467.133.8 19.8 AD-63473.1 28.9 3.4 AD-63432.1 36.5 7.4 AD-63438.1 27.3 4.3AD-63444.1 54.6 36.0 AD-63450.1 42.0 6.1 AD-63456.1 36.6 10.2 AD-63462.123.3 3.0 AD-63468.1 48.8 27.3 AD-63474.1 23.8 3.2 AD-63433.1 51.8 13.8AD-63439.1 41.7 5.5 AD-63445.1 74.6 6.1 AD-63451.1 49.6 9.0 AD-63457.126.7 4.9 AD-63463.1 27.8 3.8 AD-63469.1 48.4 14.0 AD-63475.1 40.3 1.4AD-63434.1 93.3 9.9 AD-63440.1 37.6 4.7 AD-63446.1 38.1 15.4 AD-63452.142.3 4.0 AD-63458.1 29.7 7.9 AD-63464.1 25.7 3.4 AD-63470.1 44.8 7.8AD-63476.1 33.9 4.7 AD-63435.1 23.4 5.2 AD-63441.1 37.1 4.5 AD-63447.146.5 9.0 AD-63453.1 73.1 16.8 AD-63459.1 31.8 4.6 AD-63465.1 27.3 6.6AD-63471.1 19.5 3.1 AD-63477.1 35.2 4.7 AD-63436.1 21.8 4.7 AD-63442.144.1 11.2 AD-63448.1 33.6 6.0 AD-63454.1 58.2 16.8 AD-63460.1 27.7 2.4AD-63466.1 27.1 4.4 AD-63472.1 20.5 4.1 AD-63478.1 36.3 7.3 AD-63484.148.4 31.3 AD-63490.1 44.0 6.1 AD-63496.1 45.5 19.9 AD-63502.1 49.0 18.3AD-63508.1 41.4 2.7 AD-63514.1 36.0 5.1 AD-63520.1 40.9 4.2 AD-63479.135.1 6.5 AD-63485.1 45.5 24.0 AD-63491.1 69.0 14.5 AD-63497.1 57.1 25.1AD-63503.1 36.0 15.3 AD-63509.1 29.7 6.4 AD-63515.1 33.9 5.7 AD-63521.1117.2 10.2 AD-63480.1 38.6 0.7 AD-63486.1 48.5 12.1 AD-63492.1 38.7 3.7AD-63498.1 64.6 20.3 AD-63504.1 41.7 1.9 AD-63510.1 39.6 4.0 AD-63516.130.9 4.8 AD-63522.1 56.4 15.6 AD-63481.1 72.0 7.3 AD-63487.1 128.8 48.9AD-63493.1 31.7 6.7 AD-63499.1 44.2 17.7 AD-63505.1 69.4 7.6 AD-63511.143.8 5.3 AD-63517.1 75.3 2.2 AD-63523.1 82.1 10.6 AD-63482.1 40.1 12.2AD-63488.1 42.3 12.7 AD-63494.1 19.0 1.1 AD-63500.1 30.2 11.2 AD-63506.130.5 7.6 AD-63512.1 38.1 15.2 AD-63518.1 35.0 7.3 AD-63524.1 60.5 3.7AD-63483.1 22.7 3.6 AD-63489.1 47.6 13.7 AD-63495.1 31.0 12.7 AD-63501.124.3 2.1 AD-63507.1 37.4 7.0 AD-63513.1 32.3 5.1 AD-63519.1 46.0 6.6AD-63525.1 66.5 14.5 AD-63531.1 104.0 24.1 AD-63537.1 32.1 3.4AD-63543.1 31.2 3.8 AD-63549.1 35.2 5.2 AD-63555.1 41.7 9.3 AD-63561.144.2 7.0 AD-63567.1 39.2 4.9 AD-63526.1 66.9 15.7 AD-63532.1 90.3 17.8AD-63538.1 50.8 11.5 AD-63544.1 31.9 2.4 AD-63550.1 35.0 8.8 AD-63556.131.0 6.0 AD-63562.1 20.2 2.4 AD-63568.1 30.6 2.7 AD-63527.1 28.8 2.4AD-63533.1 63.3 6.9 AD-63539.1 28.4 3.5 AD-63545.1 26.9 8.5 AD-63551.152.5 4.7 AD-63557.1 26.7 2.2 AD-63563.1 28.1 2.7 AD-63569.1 29.2 2.8AD-63528.1 52.9 9.0 AD-63534.1 42.5 6.8 AD-63540.1 50.5 10.9 AD-63546.153.6 10.5 AD-63552.1 38.8 5.0 AD-63558.1 49.3 3.0 AD-63564.1 69.2 3.1AD-63570.1 50.6 6.0 AD-63529.1 59.5 6.5 AD-63535.1 21.0 1.7 AD-63541.140.1 23.4 AD-63547.1 26.0 9.6 AD-63553.1 31.5 6.0 AD-63559.1 34.9 2.7AD-63565.1 43.3 5.3 AD-63571.1 41.6 4.4 AD-63530.1 127.6 15.0 AD-63536.138.0 16.0 AD-63542.1 48.3 8.4 AD-63548.1 41.9 7.9 AD-63554.1 88.2 15.2AD-63560.1 48.8 17.7 AD-63566.1 33.6 6.8 AD-63572.1 82.4 67.9 AD-63578.178.5 11.5 AD-63584.1 55.7 7.2 AD-63590.1 53.4 2.9 AD-63596.1 63.5 8.6AD-63602.1 49.3 3.6 AD-63608.1 29.2 4.4 AD-63614.1 30.0 7.4 AD-63573.196.1 14.7 AD-63579.1 38.1 4.5 AD-63585.1 40.0 2.1 AD-63591.1 30.5 2.5AD-63597.1 55.1 5.8 AD-63603.1 43.6 4.0 AD-63609.1 37.7 2.7 AD-63615.144.4 9.7 AD-63574.1 44.3 10.3 AD-63580.1 33.1 3.5 AD-63586.1 39.3 2.9AD-63592.1 73.7 1.6 AD-63598.1 32.4 6.6 AD-63604.1 98.7 7.1 AD-63610.142.1 7.1 AD-63616.1 55.2 10.4 AD-63575.1 27.8 3.0 AD-63581.1 36.3 3.2AD-63587.1 36.1 3.3 AD-63593.1 39.2 4.7 AD-63599.1 37.0 5.6 AD-63605.149.3 3.7 AD-63611.1 88.8 7.7 AD-63617.1 45.6 6.6 AD-63576.1 59.9 2.9AD-63582.1 82.9 8.3 AD-63588.1 33.5 6.7 AD-63594.1 64.7 18.0 AD-63600.199.5 11.9 AD-63606.1 40.8 2.7 AD-63612.1 44.5 5.3 AD-63618.1 41.7 4.6AD-63577.1 31.1 0.3 AD-63583.1 57.3 8.6 AD-63589.1 61.9 5.9 AD-63595.151.2 8.5 AD-63601.1 70.7 15.4 AD-63607.1 39.4 1.9 AD-63613.1 36.8 2.7AD-63619.1 83.8 13.8 AD-63620.1 69.4 7.3 AD-63621.1 30.6 3.1 AD-63622.151.8 8.4 AD-63623.1 37.3 8.6

1. A double stranded RNAi agent capable of inhibiting expression ofTMPRSS6 in a cell, wherein said double stranded RNAi agent comprises asense strand and an antisense strand forming a double-stranded region,wherein said sense strand comprises at least 15 contiguous nucleotidesdiffering by no more than 3 nucleotides from any one of the nucleotidesequences of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, SEQ ID NO:4, orSEQ ID NO:5, and said antisense strand comprises at least 15 contiguousnucleotides differing by no more than 3 nucleotides from any one of thenucleotide sequences of SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8, SEQ IDNO:9, or SEQ ID NO:10, wherein substantially all of the nucleotides ofsaid sense strand and substantially all of the nucleotides of saidantisense strand are modified nucleotides, and wherein said sense strandis conjugated to a ligand attached at the 3′-terminus.
 2. The doublestranded RNAi agent of claim 1, wherein all of the nucleotides of saidsense strand and all of the nucleotides of said antisense strand aremodified nucleotides.
 3. The double stranded RNAi agent of claim 1,wherein said sense strand and said antisense strand comprise a region ofcomplementarity which comprises at least 15 contiguous nucleotidesdiffering by no more than 3 nucleotides from any one of the antisensesequences listed in any one of Tables 1, 2, 4, 5, 8, 10, and
 12. 4.(canceled)
 5. The double stranded RNAi agent of any claim 1, wherein atleast one strand comprises a 3′ overhang of at least 1 nucleotide or atleast 2 nucleotide.
 6. (canceled)
 7. A double stranded RNAi agentcapable of inhibiting expression of TMPRSS6 (matriptase-2) in a cell,wherein said double stranded RNAi agent comprises a sense strandcomplementary to an antisense strand, wherein said antisense strandcomprises a region complementary to part of an mRNA encoding TMPRSS6,wherein each strand is about 14 to about 30 nucleotides in length,wherein said double stranded RNAi agent is represented by formula (III):sense: 5′n _(p)-N_(a)-(XXX)_(i)-N_(b)-YYY-N_(b)-(ZZZ)_(j)-N_(a)-n _(q)3′antisense: 3′n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′-n_(q)′5′  (III) wherein: i, j, k, and l are each independently 0 or 1; p,p′, q, and q′ are each independently 0-6; each N_(a) and N_(a)′independently represents an oligonucleotide sequence comprising 0-25nucleotides which are either modified or unmodified or combinationsthereof, each sequence comprising at least two differently modifiednucleotides; each N_(b) and N_(b)′ independently represents anoligonucleotide sequence comprising 0-10 nucleotides which are eithermodified or unmodified or combinations thereof; each n_(p), n_(p)′,n_(q), and n_(q)′, each of which may or may not be present,independently represents an overhang nucleotide; XXX, YYY, ZZZ, X′X′X′,Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of threeidentical modifications on three consecutive nucleotides; modificationson N_(b) differ from the modification on Y and modifications on N_(b)′differ from the modification on Y′; and wherein the sense strand isconjugated to at least one ligand. 8-10. (canceled)
 11. The doublestranded RNAi agent of claim 7, wherein the YYY motif occurs at or nearthe cleavage site of the sense strand. 12-17. (canceled)
 18. The doublestranded RNAi agent of claim 7, wherein the double-stranded region is15-30 nucleotide pairs in length. 19-23. (canceled)
 24. The doublestranded RNAi agent of claim 7, wherein each strand has 15-30nucleotides.
 25. (canceled)
 26. The double stranded RNAi agent of claim7, wherein the modifications on the nucleotides are selected from thegroup consisting of LNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-alkyl,2′-O-allyl, 2′-C-allyl, 2′-fluoro, 2′-deoxy, 2′-hydroxyl, andcombinations thereof.
 27. (canceled)
 28. The double stranded RNAi agentof claim 1, wherein the ligand is one or more GalNAc derivativesattached through a bivalent or trivalent branched linker.
 29. The doublestranded RNAi agent of claim 1, wherein the ligand is


30. (canceled)
 31. (canceled)
 32. The double stranded RNAi agent ofclaim 1, wherein said agent further comprises at least onephosphorothioate or methylphosphonate internucleotide linkage. 33-40.(canceled)
 41. The double stranded RNAi agent of claim 32, wherein saidRNAi agent comprises 6-8 phosphorothioate internucleotide linkages.42-52. (canceled)
 53. The double stranded RNAi agent of claim 1, whereinsaid RNAi agent is selected from the group of RNAi agents listed in anyone of Tables 1, 2, 4, 5, 8, 10, and
 12. 54. (canceled)
 55. (canceled)56. A double stranded RNAi agent capable of inhibiting expression ofTMPRSS6 in a cell, wherein said double stranded RNAi agent comprises asense strand and an antisense strand forming a double stranded region,wherein said sense strand comprises at least 15 contiguous nucleotidesdiffering by no more than 3 nucleotides from any one of the nucleotidesequences of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, SEQ ID NO:4, orSEQ ID NO:5, and said antisense strand comprises at least 15 contiguousnucleotides differing by no more than 3 nucleotides from any one of thenucleotide sequences of SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8, SEQ IDNO:9, or SEQ ID NO:10, wherein substantially all of the nucleotides ofsaid sense strand comprise a modification selected from the groupconsisting of a 2′-O-methyl modification and a 2′-fluoro modification,wherein said sense strand comprises two phosphorothioate internucleotidelinkages at the 5′-terminus, wherein substantially all of thenucleotides of said antisense strand comprise a modification selectedfrom the group consisting of a 2′-O-methyl modification and a 2′-fluoromodification, wherein said antisense strand comprises twophosphorothioate internucleotide linkages at the 5′-terminus and twophosphorothioate internucleotide linkages at the 3′-terminus, andwherein said sense strand is conjugated to one or more GalNAcderivatives attached through a branched bivalent or trivalent linker atthe 3′-terminus. 57-67. (canceled)
 68. A pharmaceutical compositioncomprising the double stranded RNAi agent of claim
 1. 69-73. (canceled)74. A method of inhibiting TMPRSS6 expression in a cell, the methodcomprising: (a) contacting the cell with the double stranded RNAi agentof claim 1; and (b) maintaining the cell produced in step (a) for a timesufficient to obtain degradation of the mRNA transcript of a TMPRSS6gene, thereby inhibiting expression of the TMPRSS6 gene in the cell.75-81. (canceled)
 82. A method of treating a subject having a TMPRSS6associated disorder, comprising administering to the subject atherapeutically effective amount of the double stranded RNAi agent ofclaim 1 or a pharmaceutical composition of claim 68, thereby treatingsaid subject. 83-100. (canceled)
 101. The double stranded RNAi agent ofclaim 7, wherein the ligand is one or more GalNAc derivatives attachedthrough a bivalent or trivalent branched linker.
 102. The doublestranded RNAi agent of claim 7, wherein the ligand is


103. The double stranded RNAi agent of claim 7, wherein said agentfurther comprises at least one phosphorothioate or methylphosphonateinternucleotide linkage.
 104. The double stranded RNAi agent of claim103, wherein said RNAi agent comprises 6-8 phosphorothioateinternucleotide linkages.
 105. The double stranded RNAi agent of claim7, wherein said RNAi agent is selected from the group of RNAi agentslisted in any one of Tables 1, 2, 4, 5, 8, 10, and 12.